Crosslinked polymer nano-assemblies and uses thereof

ABSTRACT

The invention provides a novel system of nano-assemblies and related method for delivery of therapeutic, diagnostic or imaging agent to biological sites. The compositions and methods of the invention enable the syntheses of novel polymeric nano-assemblies (nanoparticles) under non-emulsion conditions with the incorporation of hydrophobic guest molecules. The versatilities and advantages of the polymer nanoparticles of the invention include: (i) the guest molecules (e.g., drug molecules) can be readily incorporated non-covalently within the nanoparticles; (ii) the surface of the nanoparticles are functionalizable; (iii) the non-covalently encapsulated guest molecule (payload) can be released in response to a biologically relevant stimulus at the target site; (iv) the payload is held by the polymeric nanoparticle before being internalized in cells and can be released within the cellular interiors; (v) encapsulating lipophilic small molecules within its crosslinked interiors and binding proteins on its surface through electrostatic interactions; (vi) facile synthetic methods for ligand functionalization that can be utilized to decorate nanogels with cell targeting ligands that facilitate receptor-dependent cellular uptake, and (vii) the payload release kinetics is tunable and controllable.

PRIORITY CLAIMS AND CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 61/489,201, filed May 23, 2011, the entire contentof which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

The United States Government has certain rights to the inventionpursuant to Grant No. W911NF-07-1-0462 from DARPA, Grant No. DMR-0820506from the National Science Foundation (MRSEC), and Grant No. CMMI-0531171from the National Science Foundation (NSEC) to the University ofMassachusetts.

TECHNICAL FIELDS OF THE INVENTION

The invention generally relates to polymer nano-structures and drugdelivery. More particularly, the invention relates to novel,functionalized polymer nano-assemblies that are useful as drug deliveryvehicles and related methods for making the nano-assemblies and forcontrolled and targeted delivery of therapeutic or diagnostic agents.

BACKGROUND OF THE INVENTION

Major challenges remain in controlled administration of insoluble andtoxic hydrophobic drugs to target sites. Although tremendous progresshas been made in the field of delivery vehicle design, the criticalissues of encapsulation stability and versatility of the deliveryvehicles continue to present major difficulties. (Goldberg, et al. 2007J. Biomater. Sci.—Polym. E. 18, 241-268; Allen, et al. 2004 Science 303,1818-1822; Savic, et al. 2006 J. Drug. Target 14, 343-355.) A goal thatcontinues to elude researchers is a functional system wherein awater-soluble container non-covalently binds hydrophobic guest moleculesand releases them in a controlled manner in response to a specifictrigger. (Peer, et al. 2007 Nat. Nanotechnol. 2, 751-760; Haag 2004Angew. Chem. Int. Ed. 43, 278-282; Ganta, et al. 2008 J. Control.Release 126, 187-204; Allen, et al. 2004 Science 303, 1818-1822.) Whensuch a container is based on a nano-sized host, there is an even greaterinterest because of the potential in passive targeting of tumor tissuethrough the so-called enhanced permeability and retention (EPR) effect.(Maeda, et al. 2000 J. Control. Release 65, 271-284.)

Nano-scale supramolecular micellar assemblies are promising candidatesbecause they are capable of non-covalently sequestering hydrophobicguest molecules in aqueous solution. (Duncan 2003 Nat. Rev. DrugDiscovery 2, 347-360; Liu, et al. 2009 Macromolecules 42, 3-13; Davis,et al. 2008 Nat. Rev. Drug Discovery 7, 771-782; Kataoka, et al. 2001Adv. Drug Deliv. Rev. 47, 113-131; Savić, et al. 2003 Science 300,615-618.) However, micellar assemblies formed from small moleculesurfactants have inherent stability issues.

Water-soluble cross-linked polymer nanoparticles or nanogels that cansequester lipophilic guest molecules within their interiors is of greatinterest in various applications ranging from delivery vehicles fortherapeutics, to diagnostics to theranostics, among others. (Farokhzad,et al. 2004 Science 303, 1818.) However, the classical preparativemethods including microemulsion or inverse microemulsion ones do notconveniently allow the nanogels to be water-soluble and encapsulatelipophilic guest molecules simultaneously. (Bachelder, et al. 2008 J.Am. Chem. Soc. 130, 10494; Oh, et al. 2007 J. Am. Chem. Soc. 129, 5939.)

Assemblies formed from amphiphilic polymers tend to exhibit enhancedstabilities, although they face significant complications because of arequisite concentration for assembly formation, which drastically limitsthe practicality of in vivo micelle utilization. Large dilution ofinjected micelles into the body can destabilize the self-assemblingsystems and cause uncontrolled and undesirable release of theencapsulated drug payload before arrival at the target site. (Bae, etal. 2008 J. Control. Release 131, 2-4.) Moreover, the interactionbetween micelles and biological components, such as cellular membranesand blood components, can lead to premature release of the payload fromthe micelle core. (Chen, et al. 2008 Proc. Natl. Acad. Sci. U.S.A. 105,6596-6601; Chen, et al. 2008 Langmuir 24, 5213-5217.) Therefore,alternate strategies are desired to overcome such premature release.

Due to their cross-linked nature, polymer nanogels potentially provideboth high encapsulation stability and potential for triggered release.(Byrne, et al. 2002 Adv. Drug Deliv. Rev. 54, 149-161; Kabanov, et al.2009 Angew. Chem. Int. Ed. 48, 5418-5429; Kopecek 2002 Nature 417,388-391; Hamidi, et al. 2008 Adv. Drug Deliv. Rev. 60, 1638-1649.)Current synthetic methods for nanogel preparation are based onwater-in-oil emulsion, in which inverse micelles, formed fromsurfactants in non polar solvent, provide an aqueous interior as areaction template for polymerization. (Bachelder, et al. 2008 J. Am.Chem. Soc. 130, 10494-10495; Sission, et al. 2009 Angew. Chem. Int. Ed.48, 7540-7545; Kriwet, et al. 1998 J. Controlled Release 56, 149-158;Oh, et al. 2008 Prog. Polym. Sci. 33, 448-477.)

The reported nanoparticles or nanogels, however, suffer from significantlimitations as they are prepared by microemulsion or inversemicroemulsion methods. (Sisson, et al. 2009 Angew. Chem. Int. Ed. 48,7540-7545; Bachelder, et al. 2008 J. Am. Chem. Soc. 130, 10494-10495;Oh, et al. 2007 J. Am. Chem. Soc. 129, 5939-5945.) These methods arecomplex and require multiple purification steps to remove unreactedmonomers and surfactant materials that were used to stabilize theemulsion. When a water-soluble polymer nanoparticle is targeted, inversemicroemulsion-based synthesis is a preferred method. The continuousphase in the inverse microemulsion (water-in-oil emulsion) method isbased on a lipophilic solvent and, therefore, cannot be used toencapsulate hydrophobic guest molecules during nanoparticle formation.An attractive alternate to forming polymer nanoparticles is to collapsea limited number of polymer chains to achieve the desired nanoparticles.The reported methods required ultrahigh dilution conditions or inverseaddition conditions, which significantly limit the capabilities in guestmolecule incorporation. (Kadlubowski, et al. 2003 Macromolecules 36,2484-2492; Jiang, et al. 2005 Macromolecules 38, 5886-5891; Mackay, etal. 2003 Nat. Mater. 2, 762-766; Cheman, et al. 2007 J. Am. Chem. Soc.129, 11350-11351; Harth, et al. 2002 J. Am. Chem. Soc. 124, 8653-8660.)

Furthermore, nanoscale vehicles are desired that can concurrentlysequester and deliver two different molecules. (Kelkar, et al. 2011Bioconjug. Chem. 22, 1879-1903; Kelkar, et al. 2011 Acc. Chem. Res. 44,Issue #10; Jain 2001 Nature Med. 7, 987-989; Sengupta, et al. 2005Nature, 436, 568-572.) It is difficult and complicated when acombination of a water-soluble hydrophilic molecule and awater-insoluble lipophilic molecule are to be co-encapsulated anddelivered, partly due to the tendency of proteins to irreversiblydenature under non-native conditions. (Kim, et al. 2009 Langmuir 25,14086-14092; Kim, et al. 2011 Mol. Pharmaceutics. 8, 1955-1961;Wiradharma, et al. 2009 Biomaterials 30, 3100-3109.)

Targeting ligands, such as folic acid, have been studied as componentsof active targeting systems in drug delivery. (Nasongkla, et al. 2004Angew. Chem. Int. Ed. 43, 6323-6327; Lee, et al. 2008 Angew. Chem. Int.Ed., 47, 2418-2421; Aluri, et al. 2009 Adv. Drug Delivery Rev., 61,940-952; Sudimack, et al. 2000 Adv. Drug Delivery. Rev. 41, 147-161.)Ligands are attached to the hydrophilic ends of assembly-formingamphiphilic molecules. (Sutton, et al. 2007 Pharm. Res. 24, 1029-1046;Yoo, et al. 2004 J. Controlled Release 96, 273-283; Xu, et al. 2007Angew. Chem. Int. Ed. 46, 4999-5002.) However, the installation of suchligands onto these molecules requires complicated synthetic steps,limiting the versatility of ligand functionalization to tailor carriersfor targeting specific cell types.

Thus, novel and improved polymer nano-assemblies and methods ofpreparation are required to overcome the limitations and shortcomings ofthe existing methods, in particular, those that can transport diversepayloads and with versatile and effective targeting capabilities arestrongly desired.

SUMMARY OF THE INVENTION

The invention is based, in part, on the unexpected discovery of novelpolymeric nano-assemblies (nanoparticles or nanogels) and novel methodsthat allow for their syntheses under non-emulsion conditions with theincorporation of hydrophobic guest molecules. The polymer nanoparticlesof the invention offer versatilities and advantages over the existingsystems. The guest molecules (payload) can be readily incorporatednon-covalently within the nanoparticles and can be released in acontrolled manner in response to a biologically relevant stimulus at thedesired biological site. The payload is held by the polymericnanoparticle before being internalized in cells and can be releasedwithin the cellular interiors The surface of the nanoparticles arereadily functionalizable. The payload release kinetics is tunable andcontrollable allowing for diverse applications.

The invention also provides facile methods to achieve polymeric nanogelsusing simple reactions between the lipophilic activated esters anddiamines. This strategy offers the advantage that the syntheses ofnanogels are not limited to disulfide crosslinked systems orself-crosslinking reactions. The intermolecular nature of thecrosslinking reaction allows for incorporation of a broader variety ofstimuli-sensitive features in the diamine crosslinkers and thus in thenanogels.

Additionally, the invention provides nanogels that are capable ofencapsulating lipophilic small molecules within its crosslinkedinteriors and are capable of binding proteins on its surface throughelectrostatic interactions. The nanogels can be efficientlyfunctionalized with cell penetrating peptides. The nanogels bindoppositely charged proteins. The charge density on the nanogel surfaceaffects the efficiency of binding of the complementarily chargedproteins. Complexation of the protein with the nanogel does not alterthe activity of the protein, and the complex exhibits efficient uptakeby cells. Both the lipophilic small molecule and the protein areconcurrently taken up by the cells. And the enzyme retains its activityeven upon cellular entry.

Furthermore, the invention provides a facile synthetic method for ligandfunctionalization that can be utilized to decorate nanogels with celltargeting ligands. The ligand-decorated nanogels exhibit facilitatedreceptor-dependent cellular uptake. The selective internalizationcapability can be used to deliver chemotherapeutic drugs to specificreceptor-rich cells. The versatile one-pot synthetic method forsynthesizing the ligand-decorated nanogels, combined with the intrinsicencapsulation stability and targeting capabilities of the formednanogels, open up new avenues in targeted and controlled drug delivery.

In one aspect, the invention generally relates to a nano-assembly forcontrolled delivery of a therapeutic, diagnostic or imaging agent to abiological site. The nano-assembly includes: a water-soluble polymerhost comprising a network of crosslinked polymer molecules; and a guestmolecule that comprises a therapeutic, diagnostic or imaging agentnon-covalently associated with the polymer host. The therapeutic,diagnostic or imaging agent is releasable upon partial or completede-crosslinking of the crosslinked network of polymer molecules at ornear the biological site.

In another aspect, the invention generally relates to a nano-assemblyfor controlled and concurrent delivery of a small molecule agent and anon-small molecule therapeutic or diagnostic agent to a biological site.The nano-assembly includes: a water-soluble polymer host comprising acrosslinked network of polymer molecules; a guest small moleculetherapeutic, diagnostic or imaging agent non-covalently associatedwithin the crosslinked network of polymer molecules, wherein the guestsmall molecule therapeutic, diagnostic or imaging agent is releasableupon partial or complete de-crosslinking of the crosslinked network ofpolymer molecules at or near the biological site; and a guest non-smallmolecule therapeutic, diagnostic or imaging agent covalently linked toor non-covalently associated with the polymer host, wherein the guestnon-small molecule therapeutic, diagnostic or imaging agent isreleasable at or near the biological site.

In yet another aspect, the invention generally relates to a method fordelivering a therapeutic, diagnostic or imaging agent to a biologicalsite. The method includes: (1) providing a nano-assembly that includes awater-soluble polymer host comprising a crosslinked network of polymermolecules; and a guest molecule comprising a therapeutic, diagnostic orimaging agent non-covalently associated with the polymer host, whereinthe therapeutic, diagnostic or imaging agent is releasable upon partialor complete de-crosslinking of the crosslinked network of polymermolecules at or near the biological site; (2) transporting thenano-assembly to the biological site; and (3) causing de-crosslinking ofthe crosslinked network of polymer molecules, thereby releasing thetherapeutic, diagnostic or imaging agent at or near the biological site.

In certain preferred embodiments, the polymer host comprises a networkof a co-polymer (block or random) having the structural formula:

wherein

each of R₁, R′₁, and R″₁, is independently a hydrogen, C₁-C₁₂ alkylgroup, or halogen;

each of R₂, R′₂, R″₂, R₃, R′₃ and R″₃ is independently a hydrogen,(C₁-C₁₆) alkyl, (C₁-C₁₆) alkyloxy, or halogen;

each of L₁, L₂ and L₃ is independently a linking group;

each of S₁, S₂ and S₃ is independently a single bond or a spacer group;

W is a hydrophilic group;

X is a group comprising a crosslinking moiety;

Y is a non-crosslinking group; and

each of i and j is independently a positive number, k may be zero or apositive number.

In yet another aspect, the invention relates to a nano-vehicle usefulfor controlled delivery of an agent (e.g., therapeutic, diagnostic orimaging) to a biological site. The nano-vehicle includes: awater-soluble, crosslinked polymer host network; and a therapeutic,diagnostic or imaging payload non-covalently and releasably associatedwith the polymer host network. The therapeutic, diagnostic or imagingpayload is released upon partial or complete de-crosslinking of thecrosslinked polymer host network at or near the biological site.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows: (a) Exemplary structures of the polymer precursors andnanogels. (i). Cleavage of specific amount of PDS group by DTT, (ii).nanogel formation by inter/intra-chain crosslinking; (b) Schematicrepresentation of the preparation of the biodegradable nanogels.

FIG. 2 shows: Exemplary size distribution of the polymer aggregates at(a) 25° C. and (b) 70° C. in water. (c) Turbidity experiment showing thechange in high tension voltage with temperature of the polymer (Inset:magnification shows subtle changes in high tension voltage in polymers 3and 4).

FIG. 3 shows: (a) Exemplary size distribution of the nanogels preparedby DTT addition into the polymer aggregates at 25° C. or 70° C. in waterand (b) the corresponding autocorrelation functions. (c) TEM images ofthe nanogels.

FIG. 4 shows: (a) Exemplary absorption spectra of pyridothione inUV-vis. Pyridothione, which is a byproduct during nanogel synthesis bydisulfide bond formation and shows characteristic absorption at 350 nmwavelength, is monitored in each nanogel (10 mg/mL) prepared. (b) Sizedistribution of nanogels (1 mg/mL) by DLS. (c) The emission spectra ofNile red sequestered in polymer nanogels

FIG. 5 shows: Exemplary dye release from the nanogels NG1 (a, d, g), NG2(b, e, f), and NG3 (c, f, i) (0.05 wt %) in response to varied GSHconcentrations. (a-c) 10 μM GSH and (d-f) 10 mM GSH at pH 7.4, and (g-i)10 mM GSH at pH 5. The release only occurred at high GSH concentration.At acidic pH under 10 mM GSH, the release was faster and more continuousover time than that at neutral pH.

FIG. 6 shows: Exemplary comparison of GSH-induced dye release rate fromthe nanogels which have different crosslinking densities at (a) pH 7.4and (b) pH 5.

FIG. 7 shows: (a) Schematic representation of the stable and leakynanocontainers identified by a FRET experiment. (b) Fluorescence spectraof nanogels (NG3) containing a FRET pair, DiO and DiI. Nanogel solutions(2 mg/mL) were prepared containing two hydrophobic dyes, 1 wt % DiO(donor, fluorescence at 503 nm) and 1 wt % DiI (acceptor, fluorescenceat 575 nm). Time-resolved spectra of (c) NG1 (0.1 mg/mL) and (d) NG3(0.1 mg/mL) containing two dyes after mixing with 4 mM DOPC vesiclesolution. (e) Change in FRET ratio of the nanogels containing both dyes.The addition of 20 mM DTT to NG3 after 36 h showed significant decreasein the FRET ratio.

FIG. 8 shows: Exemplary confocal microscopy images of nanogelscontaining DiO and DiI as a FRET pair at different incubation times.NG1, 6% crosslinked gels, were incubated with MCF-7 cells for (a) 2 h,(b) 4 h, and (c) 24 h. NG3, 25% crosslinked gels, were incubated withMCF-7 cells for (d) 2 h, (e) 4 h, and (f) 24 h. Within each image set,top left is the DiO channel which shows green color (no FRET, dyerelease) and top right is the DiI channel which shows red color (FRET,no dye release). Bottom left is the DIC image and bottom right is anoverlap of all three. Yellow color is overlay with green and red. Scalebar is 20 μm.

FIG. 9 shows: (a) Exemplary in vitro toxicity of empty nanogels with293T cells after 24 h incubation; (b) Dox-loaded nanogels with MCF-7cells after 72 h incubation; Confocal microscopy images of (c) free-Doxafter 3 h; NG1-Dox after (d) 3 h and (e) 4 h; NG3-Dox after (f) 3 h and(g) 4 h.

FIG. 10 shows exemplary GPC traces of polymer 1-4.

FIG. 11 shows exemplary DLS of the nanogel (from polymer 2) at 25° C.and 70° C.

FIG. 12 shows exemplary TEM images of (a) NG1, (b) NG2, and (c) NG3.

FIG. 13 shows exemplary dye release from (a) NG1, (b) NG2, and (c) NG3(0.05 wt %) in 10 μM GSH at pH 5.

FIG. 14 shows exemplary DiO (green) and DiI (red) release form (a) NG1,and (b) NG3 after 48 h.

FIG. 15 shows exemplary size exclusion chromatography of Dox-containingnanogel and free Dox.

FIG. 16 shows exemplary DLS of nanogels (a) NG1, (b) NG2, and (c) NG3 atpH 7 and 5.

FIG. 17 shows a schematic illustration of an embodiment of using of thefluorescent carbocyanine dyes Dil and DiO.

FIG. 18 shows a schematic illustration of an embodiment of thenano-assemblies of the invention. (a) Schematic representation of thepreparation of bifunctional cross-linked nanogels. (b) Synthesis ofcross-linked nanogels from P1′ to make NG1′ and NG2′.

FIG. 19 shows: (a) exemplary IR spectroscopic data and (b) ¹⁹F NMRspectra of the synthesis of cross-linked polymer nanogels: reaction ofP1′ and CYS at 2 hours (black); 48 hours (red); reaction of NG2′ andpropylamine (blue).

FIG. 20 shows exemplary encapsulation stabilities of P1′ (black), NG2′(blue), and NG3′ (red) in the presence of GSH (solid line) and absenceof GSH (broken line).

FIG. 21 shows exemplary characterization of cross-linking in thepresence of hexamethylenediamine and cystamine: (a) amidation monitoredby FTIR; (b) release of pentafluorophenyl groups tracked by ¹⁹F-NMR.

FIG. 22 shows exemplary (a) Size distributions of nanogel cross-linkedby CYS, and then re-dispersed in different solvents with variousconcentrations; (b) TEM image of nanogel; The scale bar is 500 nm.

FIG. 23 shows exemplary absorbance of nanogel respectively cross-linkedby CYS and HMDA and loaded with DiI. Concentration of both measurednanogel solution is 0.2 mg/mL.

FIG. 24 shows exemplary size of nanogels prepared in a variety ofPPFPA-PEGMA concentrations.

FIG. 25 shows exemplary synthesis of nanogel in H₂O: (a) Cross-linkingfollowed by FTIR; (b) Size of nanogel prepared from 2 mg/mL, 5 mg/mL, 10mg/mL of PFPA-PEG solution in H₂O; Inset is TEM image of nanogel wherethe scale bar is 100 nm.

FIG. 26 shows exemplary charged surface of nanogels without and withpost-nanogel modification.

FIG. 27 shows exemplary size distribution of nanogel cross-linked byHMDA and dispersed in different solvents.

FIG. 28 shows exemplary FTIR spectrum of nanogel and nanogel modified byisopropylamine (IPA) and N,N-dimethylethylenediamine (DMEDA).

FIG. 29 shows exemplary end group analysis for polymer and nanogel by¹HNMR.

FIG. 30 shows exemplary nanogel-protein complexation by complementaryelectrostatic interactions.

FIG. 31 shows exemplary structures of the nanogel's polymer precursorand tri-arginine peptide.

FIG. 32 shows exemplary DLS and ζ-potential graphs of: (a) β-gal,NG1-CRRR and β-gal:NG1-CRRR complex (1:2) and (b) β-gal, NG2-CRRR andβ-gal:NG2-CRRR-β-gal complex (1:4).

FIG. 33 shows exemplary protein and hydrophobic dye delivery usingNG1-CRRR (a and c) and NG2-CRRR (b and d) without serum, and with serum,respectively. Within each image set, top left is the FITC channel whichshows green color (β-gal) and top right is the DiI channel that showsred color (hydrophobic dye). Bottom left is the DIC image and bottomright is an overlap of all three. Yellow color is overlay of green andred. Scale bar is 20 μm. (e) X-gal activity assay of delivered β-galinto cells.

FIG. 34 shows exemplary NMR spectra of PEO:PDSEMA polymer.

FIG. 35 shows exemplary NMR and Mass spectra of triarginine peptide.

FIG. 36 shows exemplary absorbance of: (a) NG1 (10 mol % DTT) and (b)NG2 (20 mol % DTT) before and after modification with CRRR peptide.

FIG. 37 shows exemplary fluorescence emission change of FITC labeledβ-gal and encapsulated DiI in (a) NG1-CRRR and (b) NG2-CRRR varyingnanogel ratios.

FIG. 38 shows exemplary Agarose Gel Electrophoresis for β-gal complexedto NG1-CRRR or NG2-CRRR.

FIG. 39 shows exemplary activity assay of β-gal complexed to NG1-CRRRand NG2-CRRR.

FIG. 40 shows exemplary intracellular delivery of native β-gal is notefficient without nanogel.

FIG. 41 shows exemplary nanogels are non-leaky, but exhibit slow celluarinternalization; polymeric micelles exhibit rapid internalizations oflipophilic drug molecules due to leakage of free drug from the assemblyinterior; T-NGs exhibit high encapsulation stability and fast cellularinternalization, which has a great potential for passive and activetargeting drug delivery.

FIG. 42 shows exemplary (a) Schematic representation of the one-potsynthesis of T-NGs, (b) chemical structure of polymer and T-NGs, and (c)cysteine-containing targeting ligands.

FIG. 43 shows exemplary (a) Dynamic light scattering of polymeraggregates (10 mg/mL) at 60° C. and T-NG (10 mg/mL) at room temperature.UV absorbance of pyridothione (b) during nanogel synthesis (0.04 mg/mL)and (c) ligand modification with CRRR (0.05 mg/mL). (d) Zeta potentialof nanogel (NG) (1 mg/mL) and NG-RRR (1 mg/mL). (e) AFM image of T-NG (2μm×2 μm).

FIG. 44 shows exemplary fluorescence emission spectra of mixed polymeraggregates (a) and NG (b) separately encapsulated DiI/DiO.

FIG. 45 shows exemplary cell internalization of DiO which isencapsulated in polymer aggregates (a) and NG (b) at different celllines (left: 293T, middle: MCF7, right: HeLa, scale bar=10 μm). (c) Insitu cell internalization of hydrophobic dye by FRET (left: DiO channel,middle: DiI channel, right: overlap of both channel). No FRET insidecell indicates that the dyes only were transferred into the cellswithout polymer aggregates. Each cell line was cultured in DMEM/F12 with10% FBS supplement.

FIG. 46 shows exemplary confocal microscopy images of T-NGs containingDiO at different cell lines, (a) 293T, (b) MCF7, (c) HeLa, and (d)SKOV3. (left: NG-RRR, middle: NG-FA, right: NG-RGD, scale bar=10 μm).Each cell line was cultured in DMEM/F12 with 10% FBS supplement.

FIG. 47 shows exemplary in vitro toxicity of PTX-loaded NG-RGD with MCF7and HeLa cells after 72 h incubation. The loading amount of PTX is 5 wt% for polymer and NG. 10 μM of PTX is used for all experiments.

FIG. 48 shows exemplary ¹H-NMR and MS of cell penetrating peptide CRRR.

FIG. 49 shows exemplary ¹H-NMR and MS of cysteine-folic acid.

FIG. 50 shows exemplary (a) Turbidity experiment shows the change inhigh tension voltage with temperature of the polymer due to the LCSTbehavior of oligoethylene glycol (OEG) in the polymer side chain. b)Plot of the absorption maximum of pyridothione which is a byproduct ofthe disulfide bond formation during nanogel synthesis. The production ofpyridinethione plateaus within 20 min, indicating that disulfidecrosslinking is finished within 20 min. c) Plot of fluorescence ofresonance energy transfer (FRET) ratio vs. time. The nanogels showed nosignificant FRET ratio change, indicating the high encapsulationstability, while polymer aggregates themselves showed very fast FRETevolution, suggesting the leakage property of these aggregates.

FIG. 51 shows exemplary UV-vis absorbance of T-NGs. (a) UV-visabsorbance of pyridothione after nanogel synthesis (black) and ligandmodification (red) with CRGD (0.05 mg/mL). (b) UV-vis absorbance offolic-acid decorated (0.05 mg/mL). The measurement was performed afterpurification by dialysis because the absorbance of folic acid andpyridothione are overlapped. (c) UV-vis absorbance of pyridothione oftaxol containing nanogel (black) and ligand modification (red) with CRGD(0.05 mg/mL).

FIG. 52 shows exemplary (a) In vitro toxicity of polymer, emptynanogels, and RGD-coated nanogels with 293T cells after 24 h incubation.Cell viability was measured using the Alamar Blue assay. The polymer,empty nanogels, and RGD-coated nanogels exhibit high cell viability andno concentration dependent toxicity up to the concentration of 0.5mg/mL, indicating that the all material is non-toxic.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. General principles of organic chemistry,as well as specific functional moieties and reactivity, are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999. It will be appreciated that the compounds, as describedherein, may be substituted with any number of substituents or functionalmoieties.

As used herein, (C_(x)-C_(y)) refers in general to groups that have fromx to y (inclusive) carbon atoms. Therefore, for example, C₁-C₆ refers togroups that have 1, 2, 3, 4, 5, or 6 carbon atoms, which encompassC₁-C₂, C₁-C₃, C₁-C₄, C₁-C₅, C₂-C₃, C₂-C₄, C₂-C₅, C₂-C₆, and all likecombinations. (C₁-C₂₀) and the likes similarly encompass the variouscombinations between 1 and 20 (inclusive) carbon atoms, such as (C₁-C₆),(C₁-C₁₂) and (C₃-C₁₂).

As used herein, the term “(C_(x)-C_(y))alkyl” refers to a saturatedlinear or branched free radical consisting essentially of x to y carbonatoms, wherein x is an integer from 1 to about 10 and y is an integerfrom about 2 to about 20. Exemplary (C_(x)-C_(y))alkyl groups include“(C₁-C₂₀)alkyl,” which refers to a saturated linear or branched freeradical consisting essentially of 1 to 20 carbon atoms and acorresponding number of hydrogen atoms. Exemplary (C₁-C₂₀)alkyl groupsinclude methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,dodecanyl, etc.

As used herein, the term, “(C_(x)-C_(y))alkoxy” refers to a straight orbranched chain alkyl group consisting essentially of from x to y carbonatoms that is attached to the main structure via an oxygen atom, whereinx is an integer from 1 to about 10 and y is an integer from about 2 toabout 20. For example, “(C₁-C₂₀)alkoxy” refers to a straight or branchedchain alkyl group having 1-20 carbon atoms that is attached to the mainstructure via an oxygen atom, thus having the general formula alkyl-O—,such as, for example, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy,sec-butoxy, tert-butoxy, pentoxy, 2-pentyl, isopentoxy, neopentoxy,hexoxy, 2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

As used herein, the term “halogen” refers to fluorine, chlorine,bromine, or iodine.

DETAILED DESCRIPTION OF THE INVENTION

The invention generally provides nano-scale assemblies as drug deliveryvehicles that encapsulate drug molecules as payloads and release them inresponse to a pre-selected and specific trigger. Delivery systems of theinvention are of great interest in therapeutic applications, especiallyfor cancer, arthritis and related diseases with inflamed organs,tissues, or cells.

Micellar systems, due to their capacity to bind hydrophobic guestmolecules non-covalently, are potentially good delivery vehicles toovercome the bottlenecks of traditional chemotherapies. However, thedrug encapsulation stability of self-assembled systems during bloodcirculation continues to be a major obstacle. Thus, deliberate moleculardesign is required for stable encapsulation, targeting and triggeredrelease.

Crosslinked polymer nanogels are promising systems for non-covalentlyencapsulating and delivering active drug molecules. However, significanthurdles remain because the classical preparative methods for polymernanogels do not facilitate the nanogels to be both water-soluble andencapsulate lipophilic guest molecules simultaneously. These two areessential elements of a nanoscopic delivery vehicle.

The invention provides novel polymeric nano-assemblies (nanoparticles ornanogels) and novel methods of syntheses under non-emulsion conditionswith the incorporation of hydrophobic guest molecules. The guestmolecules (payload) can be readily incorporated non-covalently withinthe nanoparticles. The payload is held by the polymeric nanoparticlebefore being internalized and released within the cellular interiors ina controlled manner in response to a biologically relevant stimulus atthe desired biological site. The payload release kinetics is tunable andcontrollable allowing for diverse applications. The surface of thenanoparticles is readily functionalizable.

The invention also provides facile methods to achieve polymeric nanogelsusing simple reactions between the lipophilic activated PFP ester anddiamines. The syntheses of nanogels are not limited to disulfidecrosslinked systems or self-crosslinking reactions. The intermolecularnature of the crosslinking reaction allows for incorporating a broadervariety of stimuli-sensitive features in the diamine crosslinkers andthus in the nanogel. A key feature is that a lipophilic functional groupthat is reactive to a specific functional group is utilized to obtainthe nano-assembly, which can then be locked into a nanogel by triggeringa reaction.

In addition, the invention provides nanogels that is capable ofencapsulating lipophilic small molecules within its crosslinkedinteriors and binding proteins on its surface through electrostaticinteractions. The nanogels can be functionalized with cell penetratingpeptides efficiently. The nanogels bind oppositely charged proteins andthat the charge density on the nanogel surface affects the efficiency ofbinding of the complementarily charged proteins. Complexation of theprotein with the nanogel does not alter the activity of the protein, andboth the lipophilic small molecule and the protein are concurrentlytaken up by cells. The enzymatic activity remains intact even uponcellular entry. Such design strategy outlined herein can have beenapplied in a variety of areas including therapeutics, diagnostics, and acombination of the two by way of nanotheranostics.

Furthermore, ligand functionalization can be facilely achieved that canbe utilized to decorate nanogels with cell targeting ligands. Theligand-decorated nanogels exhibit facilitated receptor-dependentcellular uptake. The selective internalization capability can be used todelivering chemotherapeutic drugs to a specific receptor-rich cells. Theversatile one-pot synthetic method for synthesizing the ligand-decoratednanogels, combined with the intrinsic encapsulation stability andtargeting capabilities of the formed T-NGs, open up new avenues intargeted drug delivery for crosslinked polymer nanogels.

In one aspect, the invention generally relates to a nano-assembly forcontrolled delivery of a therapeutic, diagnostic or imaging agent to abiological site. The nano-assembly includes: a water-soluble polymerhost comprising a network of crosslinked polymer molecules; and a guestmolecule that comprises a therapeutic, diagnostic or imaging agentnon-covalently associated with the polymer host. The therapeutic,diagnostic or imaging agent is releasable upon partial or completede-crosslinking of the crosslinked network of polymer molecules at ornear the biological site.

In another aspect, the invention generally relates to a nano-assemblyfor controlled concurrent delivery of a small molecule and non-smallmolecule therapeutic, diagnostic or imaging agents to a biological site.The nano-assembly includes: a water-soluble polymer host comprising acrosslinked network of polymer molecules; a guest small moleculetherapeutic, diagnostic or imaging agent non-covalently associatedwithin the crosslinked network of polymer molecules, wherein guest smallmolecule therapeutic, diagnostic or imaging agent is releasable uponpartial or complete de-crosslinking of the crosslinked network ofpolymer molecules at or near the biological site; and a guest non-smallmolecule therapeutic, diagnostic or imaging agent covalently linked toor non-covalently associated with the polymer host, wherein the guestnon-small molecule therapeutic, diagnostic or imaging agent isreleasable at or near the biological site.

In yet another aspect, the invention generally relates to a method fordelivering a therapeutic, diagnostic or imaging agent to a biologicalsite. The method includes: (1) providing a nano-assembly that includes awater-soluble polymer host comprising a crosslinked network of polymermolecules; and a guest molecule comprising a therapeutic, diagnostic orimaging agent non-covalently associated with the polymer host, whereinthe therapeutic, diagnostic or imaging agent is releasable upon partialor complete de-crosslinking of the crosslinked network of polymermolecules at or near the biological site; (2) transporting thenano-assembly to the biological site; and (3) causing de-crosslinking ofthe crosslinked network of polymer molecules, thereby releasing thetherapeutic, diagnostic or imaging agent at the biological site.

In some embodiments, the nano-assembly further comprises targetingmoieties covalently linked to or non-covalently associated with thepolymer host. For example, the targeting moiety may be an antibody, anaptamer, a peptide, or a small molecule ligand.

Depending on the desired targeting approach of the nano-assembly, oneconsideration is that the advantage of the nano-assemblies of theinvention over small molecules is targeting by EPR effect based on sizeof the particles. The size of the nano-assembly may be designedaccording to the particular applications, for example, from about 3 nmto about 300 nm (e.g., from about 3 nm to about 200 nm, from about 3 nmto about 150 nm, from about 3 nm to about 100 nm, from about 5 nm toabout 300 nm, from about 10 nm to about 300 nm, from about 15 nm toabout 250 nm, from about 20 nm to about 250 nm, from about 30 nm toabout 250 nm, from about 50 nm to about 250 nm, from about 100 nm toabout 250 nm).

In certain preferred embodiments, the guest molecule is hydrophobic.

In certain preferred embodiments, the nano-assembly is capable ofpenetrating cellular membrane.

In some embodiments, the polymer host comprises a network of co-polymermolecules, for example, random co-polymers.

In some embodiments, the co-polymer molecules comprise pendanthydrophilic side chains. In certain preferred embodiments, the polymerhost is covalently attached to a

(or oligoethylene-glycol “OEG’) group, wherein p is an integer fromabout 5 to about 200 (e.g., from about 5 to about 150, from about 5 toabout 100, from about 5 to about 50, from about 10 to about 200, fromabout 20 to about 200, from about 50 to about 200, from about 100 toabout 200, from about 10 to about 30, from about 10 to about 50).

In certain preferred embodiments, the polymer host comprises a networkof a co-polymer (e.g., block or random) having the structural formula:

wherein

each of R₁, R′₁ and R″₁ is independently a hydrogen, C₁-C₁₂ alkyl group,or halogen;

each of R₂, R′₂, R″₂, R₃, R′₃ and R″₃ is independently a hydrogen,(C₁-C₁₆) alkyl, (C₁-C₁₆) alkyloxy, or halogen;

each of L₁, L₂ and L₃ is independently a linking group;

each of S₁, S₂ and S₃ is independently a single bond or a spacer group;

W is a hydrophilic group;

X is a group comprising a crosslinking moiety;

Y is a non-crosslinking group; and

each of i and j is independently a positive number, k may be zero or apositive number.

In certain preferred embodiments, each of R₂, R′₂, R″₂, R₃, R′₃ and R″₃is a hydrogen, and each of R₁, R′₁ and R″₁ is a methyl group. In someembodiments, at least one of R₁, R′₁ and R″₁ is a not methyl group.

In some embodiments, each of L₁, L₂ and L₃ is independently a group of

In certain preferred embodiments, W is

wherein p is an integer from about 1 to about 20 (e.g., from about 1 toabout 3, from about 1 to about 6, from about 1 to about 9, from about 1to about 12).

W may be a charged group, for example, an anionic or cationic group suchas —CO₂ ⁻, —HPO₃ ⁻, —SO₃ ⁻, —NR₂, —NR₃ ⁺, wherein R is hydrogen or aC₁-C₂₀ alkyl group (e.g., a methyl, an ethyl, a C₁-C₃ alkyl, a C₁-C₆alkyl, a C₁-C₉ alkyl group, a C₁-C₁₂ alkyl group).

W is a zwitterionic group, for example:

wherein each R is hydrogen or a C₁-C₂₀ alkyl group (e.g., a methyl, aethyl, a C₁-C₃ alkyl, a C₁-C₆ alkyl, a C₁-C₉ alkyl group, a C₁-C₁₂ alkylgroup; n is independently an integer from about 1 to about 12 (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12).

W may also be a charge-neutral group, for instance:

In certain preferred embodiments, the polymer host comprises a networkof a co-polymer (e.g., block or random) having the structural formula:

wherein p is an integer from about 1 to about 20 (e.g., from about 1 toabout 3, from about 1 to about 6, from about 1 to about 9, from about 1to about 12).

In certain preferred embodiments, X includes a disulfide group. Anexample of stimulus-responsive functional groups is a disulfide bond,which is susceptible to the biochemical reductants such as glutathione,thioredoxin, and peroxiredoxin. (Bernkop-Schniirch, et al. 2005 Adv.Drug Deliv. Rev. 57, 1569-1582; Yang, et al. 2006 Proc. Natl. Acad. Sci.U.S.A. 103, 13872-13877; Bauhuber, et al. 2009 Adv. Mater. 21,3286-3306.)

In some embodiments, X includes a

group, wherein each of R₄ and R′₄ is independently a hydrogen or C₁-C₁₂alkyl (e.g., methyl, ethyl, a C₃-C₆ alkyl) group and X_(L) is a spacergroup. In certain preferred embodiments, each of R₄ and R′₄ is hydrogen.

X_(L) may be a pH-sensitive functional group, for example:

wherein R is hydrogen, a C₁-C₂₀ alkyl group, or a

group, wherein p is from about 1 to about 100 (e.g., from about 1 toabout 10, from about 1 to about 20, from about 1 to about 30, from about1 to about 40, from about 1 to about 50).

In some embodiments, X_(L) is a peptide having from about 1 to about 20amino acid units (e.g., from about 1 to about 6, from about 1 to about9, from about 1 to about 12) that are cleavable by an enzyme.

Y may be a linear or branched C₁-C₂₀ alkyl chain containing an aromaticmoiety. This moiety may also be derived from an amino acid phenylalanine or histidine, for example.

In some embodiments, Y includes a folic acid moiety, such as:

wherein q is an integer from about 1 to about 20 (e.g., from about 1 toabout 3, from about 1 to about 6, from about 1 to about 9, from about 1to about 12).

In some embodiments, Y comprises an imidazole moiety such as:

Syntheses of the nano-assemblies of the invention can be readilyachieved in aqueous phase from a water-soluble precursor polymer.Incorporation of surface functionalities and non-covalent encapsulationof hydrophobic guest molecules can be achieved under mild conditions. Insome embodiments, the co-polymer has a molecular weight from about 1,000to about 100,000 (e.g., from about 2,000 to about 100,000, from about5,000 to about 100,000, from about 10,000 to about 100,000, from about20,000 to about 100,000, from about 50,000 to about 100,000, from about1,000 to about 10,000, from about 1,000 to about 10,000, from about1,000 to about 20,000, from about 1,000 to about 50,000).

In some embodiments, the ratio of i:j is in the range from about 2:8 toabout 8:2 (e.g., from about 3:7 to about 7:3, from about 4:6 to about6:4, about 1:1).

In some embodiments, where from about 1% to about 95% of j issubstituted by k, for example, from about 2% to about 95%, from about 5%to about 95%, from about 10% to about 95%, from about 20% to about 95%,from about 30% to about 95%, from about 40% to about 95%, from about 1%to about 90%, from about 1% to about 85%, from about 1% to about 80%,from about 1% to about 75%, from about 1% to about 70%, from about 1% toabout 60%.

The hydrophilic-lipophilic balance (HLB) has a significant impact on theaqueous phase solubility as well as the encapsulation (loading andstability) and release of a lipophilic drug from the nano-assemblies.For some applications, one may simply vary the HLB of the nano-particlesby increasing OEG-based monothiol or an alkanethiol to increase thehydrophilicity or the lipophilicity of the gel respectively. Thisapproach also provides an added advantage: By converting any residualPDS groups to vary the HLB, one mitigates toxicity and concurrentlyoptimizes drug loading capacity, encapsulation stability, and release.

The crosslinked network of polymer molecules may be crosslinkedinter-molecularly, intra-molecularly, or both. In certain preferredembodiments, the crosslinked polymer molecules are crosslinked viadisulfide bonds.

The crosslinked network of polymer molecules may have a crosslinkingdensity from about 2% to about 80%, relative to the total number ofstructural units in the polymer. For example, the crosslinked network ofpolymer molecules may have a crosslinking density from about 2% to about70%, from about 2% to about 60%, from about 2% to about 50%, from about2% to about 40%, from about 2% to about 30%, from about 2% to about 20%,from about 2% to about 10%, from about 5% to about 80%, from about 10%to about 80%, from about 20% to about 80%, from about 30% to about 80%,from about 40% to about 80%, relative to the total number of structuralunits in the polymer.

In certain preferred embodiments, the therapeutic agent is an anti-tumoragent, for example, an anti-tumor agent is selected from doxorubicin,paclitaxel, camptothecin, rapamycin, and related chemotherapeutic drugs.

The loading weight percentage of the guest molecule may be from about0.5% to about 70% (e.g., from about 0.5% to about 60%, from about 0.5%to about 50%, from about 0.5% to about 40%, from about 0.5% to about30%, from about 1% to about 70%, from about 2% to about 70%, from about5% to about 70%, from about 10% to about 70%, from about 20% to about70%, from about 30% to about 70%).

In certain preferred embodiments, the de-crosslinking of the crosslinkednetwork of polymer molecules is caused by a biological or chemicalstimulus at the biological site. For example, the stimulus is a pH valueat the biological site or an external force such as light.

The biological site may be present on the surface of or within an organor tissue, such as lungs or tumor tissue, of a subject.

The biological site may be on the surface of or inside a cell, such asthe interstitial spaces or the interiors of a tumor cell of a subject.

In yet another aspect, the invention relates to a nano-vehicle usefulfor controlled delivery of an agent (e.g., therapeutic, diagnostic orimaging) to a biological site. The nano-vehicle includes: awater-soluble, crosslinked polymer host network; and a therapeutic,diagnostic or imaging payload non-covalently and releasably associatedwith the polymer host network. The therapeutic, diagnostic or imagingpayload is released upon partial or complete de-crosslinking of thecrosslinked polymer host network at or near the biological site.

In yet another aspect, the invention generally relates to a method formaking a polymeric nano-assembly comprising a crosslinked polymeric hostnetwork while incorporating a hydrophobic payload under non-emulsionconditions.

In certain preferred embodiments, the crosslinked polymer host networkis capable of crossing cellular membrane.

In certain preferred embodiments, the nano-vehicle further includes aguiding (e.g., targeting) moiety that is capable of bringing ortransporting the nano-vehicle to the vicinity of or inside thebiological site, wherein the guiding moiety is covalently bound to thepolymer host network.

In certain preferred embodiments, the guiding moiety is an antibody oran antibody fragment.

In certain preferred embodiments, the payload is an anti-tumor agentsuch as doxorubicin, paclitaxel, camptothecin, rapamycin, and relatedchemotherapeutic drugs.

Polymeric nano-assemblies disclosed herein are highly stable, thesurface of which can be functionalized using a simple intra/inter-chaincrosslinking reaction. The non-covalently encapsulated guest moleculescan be released in response to a certain trigger, such as a redoxtrigger, glutathione. Tunability in the release kinetics of the guestmolecules has been demonstrated through in vitro fluorescence resonanceenergy transfer (FRET) experiments.

Any suitable therapeutic, diagnostic or imaging agents may be employedaccording to the compositions, systems and methods disclosed herein.

The kinetics of drug release can be controlled, for example, by thedegree of crosslinking and by tuning the HLB. Since both nanogelformation and surface modification are accomplished by simplethiol-disulfide exchange or thiol reshuffling reactions, the reactionconditions are simple, mild, and do not require the use of organicsolvents, metal containing catalysts, or additional reagents.

The invention provides a simple, emulsion-free method for thepreparation of biocompatible nano-assemblies was developed that providesthe ability to encapsulate hydrophobic guest molecules usingintra/intermolecular disulfide formation of PDS containing polymers.Since disulfide bonds are degradable in a reducing environment, thesenanoparticles hold great potential as intracelluar drug deliveryvehicles. The release of guest molecules can be induced by externalstimuli (e.g. Redox and pH) and in vitro release rate is tunable tocrosslinking density. Additionally, it is demonstrated that thesenanoparticles can be efficiently surface-functionalized under mildconditions. The nanogels also exhibit significant stability, allowingfor tunable controlled release after cellular penetration; thesevehicles also do not seem to suffer from guest loss prior to cellularentry. Taken together, the reversible nanogel formation usingself-crosslinking polymers and corresponding method of surfacemodification open a new avenue for enhanced cytosolic drug delivery andestablish a novel approach to creating polymer nanogels for a range ofbiomedical applications from drug delivery to biosensing.

EXAMPLES

Design, Syntheses, & the Size-control of the Nanogels

Illustrative examples of structures of the nanogel precursors, polymernanogels and synthetic approach are shown in FIG. 1. The polymer nanogelprecursor is based on a random copolymer that containsoligoethyleneglycol (OEG) units and pyridyldisulfide (PDS) groups asside chain functionalities. Polymers (1-4) were prepared by reversibleaddition fragmentation chain transfer (RAFT) polymerization. The OEGunit is used to introduce a charge-neutral hydrophilic functional group,which is known to endow biocompatibility. PDS's lipophilic functionalityprovides a supramolecular amphiphilic nano-assembly in the aqueousphase, which helps avoid the use of any additional surfactant moleculesto generate the nanogel. The amphiphilic nature of the nano-assembly andlipophilic environment afforded by the PDS functionality also providesthe opportunity for lipophilic guest molecules to be sequestered withinthese nano-assemblies prior to crosslinking. The PDS functionality isreactive, but specific, to thiols and provides a mild method fordisulfide crosslinking to form the nanogel. Furthermore, since thenanogels are based on disulfide crosslinkers that can be cleaved bythiol-disulfide exchange reactions, these nanogels also have a pathwayto release the stably encapsulated guest molecules.

Four polymers were prepared that have different molecular weights anddifferent ratios of the OEG units to the PDS groups. Polymers 1 (M_(n):6,000, PDI: 1.2) and 2 (M_(n): 12,000, PDI: 1.2) have 50% of the OEGmethacrylate and 50% of the PDS-derived methacrylate. Polymers 3 (M_(n):14,000, PDI: 1.3) and 4 (M_(n): 24,000, PDI: 1.6) have 30% of the OEGmethacrylate and 70% of the PDS-derived methacrylate. The aggregatesizes of the polymers in the aqueous phase were investigated by dynamiclight scattering (DLS). Polymers 1, 2, 3, and 4 (1 wt %) showedassemblies of 5, 8, 12, and 120 nm diameter respectively in water (FIG.2a ).

The polymers showed larger aggregates at high temperature, presumablybecause of the lower critical solution temperature (LCST) behavior ofthe OEG units (FIG. 2b ). (Saeki, et al. 1976 Polymer 17, 685-689; Lutz,et al. 2006 J. Am. Chem. Soc. 128, 13046-13047; Lutz, et al. 2007Macromolecules 40, 2503-2508; Aathimanikandan, et al. 2005 J. Am. Chem.Soc. 127, 14922-14929.) The degree of hydration of multiple OEG sidechains reduces with increasing temperature resulting in a compact coilconformation and the polymer becomes more hydrophobic. This leads tointermolecular associations between the polymers, resulting in theformation of larger aggregates. (Vo, et al. 2002 Colloid. Polym. Sci.280, 400-409.)

The LCST behavior of the polymers was investigated bytemperature-dependent turbidity measurements using circular dichroism.The polymer (10 mg/mL) solutions were prepared and the changes in thehigh tension voltage were monitored at 600 nm by varying the temperatureby 2° C./min. (Klaikherd, et al. 2009 J. Am. Chem. Soc. 131, 4830-4838.)As shown in FIG. 2c , polymers 1 and 2 showed large turbidity changeabove 60° C. and 55° C., while polymers 3 and 4 showed a small changeabove 30° C. and 25° C., respectively. This observation is attributed tothe fact that the hydrophobicity in the polymer affects the LCSTbehavior. (Wang, et al. 2009 Macromolecules 42, 3026-3032.) At the sameco-monomer composition, the turbidity change and the size are largerwith increasing molecular weight due to the cooperative effect of thelarger amount of OEG units. The polymer aggregates formed using theseLCST behaviors are summarized in Table 1.

The next step involves the conversion of these polymeric aggregates intochemically crosslinked nanogels. Addition of a deficient amount ofdithiothreitol (DTT) causes the cleavage of a well-defined percentage ofthe PDS groups to the corresponding thiol functionalities. These thiolfunctionalities then react within the polymeric aggregates withunreacted PDS functionalities. This reaction results in disulfidecrosslinks within the polymeric aggregates causing the formation of thenanogels, as shown in FIG. 1.

By the addition of a deficient amount of DTT into these polymer assemblysolutions, precise control of the size of the crosslinked nanoparticlesfrom ˜10 nm to ˜200 nm in diameter was achieved. To crosslink thepolymer aggregates at different temperatures, 20 mol % of DTT againsttotal PDS groups in each polymer was added. The structures obtained fromthese reactions were characterized by transmission electron microscopy(TEM) and DLS. From polymers 1 and 2 70 nm and 100 nm nanogels wereprepared at 70° C., respectively. While the size of the polymeraggregate is reversibly sensitive to temperature, the size of thenanogel formed after the DTT-reaction retains the size observed underreaction conditions when it was cooled down to room temperature (FIG.11). This result suggests that stable crosslinked nanogels were formed,not polymer aggregates. Using polymer 3 10 nm and 26 nm nanogels wereprepared at 25° C. and 70° C., respectively, while the aggregates ofpolymer 4 were converted to nanogels of 190 nm size at 25° C. As shownin FIGS. 3a and 3b , the hydrodynamic diameter and the autocorrelationfunction in DLS reveal that fine control over the size of the nanogelscan be achieved by controlling the size of the preformed polymeraggregates in water by varying molecular weight, comonomer composition,and temperature. The evidence for the precise size control was furtherprovided by TEM experiments (FIG. 3c ). TEM images of all nanogelsrevealed well-defined spherical structures with sizes that correlatevery well with the DLS results. These results show that the nanogel sizecan be systematically tuned by controlling the structure of theprecursor polymer and by exploiting temperature-dependent aggregationthough the LCST behavior of the polymers.

TABLE 1 Properties of polymers and the sizes of the polymer aggregatesand nanogels Comonomer Aggregate Nanogel Size composition size (nm)^(c)(nm) Polymer M_(n) (PDI)^(a) (OEG:PDS)^(b) 25° C. 70° C. (PDI) 1  6900(1.2) 47:53 5 74  68 (0.07)^(d) 2 13100 (1.2) 50:50 8 142 106 (0.03)^(d)3 14400 (1.6) 33:67 12 24   26 (0.24),^(d)  10 (0.33)^(e) 4 24700 (1.6)31:69 120 255 190 (0.24)^(e) ^(a)Estimated by GPC (THF) using PMMAstandard. ^(b)Determined by NMR. ^(c)Determined by DLS. ^(d)Prepared at70° C. ^(e)Prepared at 25° C.Guest Encapsulation and Triggered/Controlled Release

Three different crosslinked particles were prepared by adding 10, 20 or50 mol % (against the precursor PDS groups) of DTT to polymer 4. Theprogress of the reaction was conveniently monitored by release of thepyridothione byproduct through tracing its characteristic absorption at343 nm. Considering the mechanism by which this addition results incrosslinked polymer particles and the percentage of PDS functionalitiesin polymer 4, this reaction should result in nanoparticles NG1, NG2, andNG3 with 7%, 14%, and 35% crosslinking densities respectively, assuming100% reaction efficiency. Estimations, based on pyridothione release,indicate that the actual crosslinking densities correspond to 6%, 13%,and 25% respectively (FIG. 4a ). DLS studies reveal that the structuresobtained are all about 190 nm in size (FIG. 4b ). TEM images revealwell-defined spherical structures with slightly smaller diameters thanthose observed in DLS, which is attributed to the possible swelling ofthe nanoparticles in water (FIG. 12). The sizes of all three nanogelsare very similar, which suggests that the size of the assembly prior tothe crosslinking reaction dictates the nanogel size and that furthercrosslinking occurs within that nanoassembly.

To investigate encapsulation of hydrophobic guest molecules within theinteriors of these nanogels, the DTT-based crosslinking reaction wascarried out in the presence of Nile red, a hydrophobic dye. Nile red isinherently insoluble in water. Therefore, the reaction was optimizedusing acetone as a solvent in the first steps, before addition of waterduring the crosslinking reaction. Isolation of the nanoparticles andtheir subsequent dissolution in water retains the presence of Nile red,as discerned by the emission spectra of all three gels (FIG. 4c ).

To test triggered release, GSH was added into nanogel solutions and therelease of Nile red was investigated by tracing the decrease in thehydrophobic dye's spectral emission intensity caused by its insolubilityin the aqueous media. To examine the GSH-dependent dye release, Nile redloaded nanogel solutions (0.05 wt %) in pH 7.4 sodium acetate buffersolution were treated with different concentrations of GSH (10 nM and 10mM) and the intensity of Nile red emission at 610 nm was monitored forthree days. At low GSH concentrations (10 nM), little dye release wasobserved for all nanogels (FIG. 5a-c ). This concentration correspondsto that commonly observed outside the cell and within the blood plasma.In contrast, high concentrations of GSH (10 mM), corresponding to thosefound inside the cell, induced significant dye release (FIG. 5d-f ).

The rate of dye release from the nanogel interior is influenced bycrosslinking density. NG1 (6% crosslinked nanogel) showed rapid releasereaching a plateau after 6 h at 10 mM GSH in pH 7.4 buffer solution. NG2(13% crosslinked nanogel) showed slower release, reaching a maximum at12 h; NG3 (25% crosslinked nanogel) displayed gradual, highly sustainedrelease for several days. Since the entry of these nanogels would likelyinvolve endocytosis, the release profile at lower pH was analyzed. Therelease profile difference under acidic conditions (pH 5) was verysimilar to that observed with pH 7 (FIG. 5g-i and FIG. 13). While thisbodes well for endocytosis based entry into the cells, the releaseprofile at high GSH concentration was surprising, as GSH activity isconsidered most efficient at neutral pH. (Moskaug, et al. 1987 J. Biol.Chem. 262, 10339-10345.) As shown in FIG. 6, similar release wasobserved for all three gels over several hours at both pH 5 and 7.4.

Encapsulation Stability and Tunable in Vitro Guest Release

The encapsulation stability of lipophilic molecules is critical to aneffective nanocarrier, preventing significant loss of drug due toleakage during circulation. A FRET based method was developed toevaluate the encapsulation stability of nanocarriers in aqueoussolutions. (Jiwpanich, et al. 2010 J. Am. Chem. Soc. 132, 10683-10685.)The crosslinking densities can significantly influence the encapsulationstability coefficient (Λ).

To correlate with the in vitro guest release experiments, FRETexperiments were carried out to investigate the dye leakage in thepresence of dioleoyl phosphatidylcholine (DOPC) bilayer vesicles. (Chen,et al. 2008 Proc. Natl. Acad. Sci. U.S.A. 105, 6596-6601.) FRET was usedbetween two non-covalently encapsulated dyes as a diagnostic tool (FIG.7a ). Nanogel solutions containing a mixture of two hydrophobic dyes,3,3′-dioctadecyloxacarbocyanine perchlorate (DiO: donor, greenfluorescence) and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanineperchlorate (DiI: acceptor, red fluorescence), were prepared. To confirmthat these two dyes exhibit the requisite FRET inside the nanogels, thenanogel solution was excited at 484 nm, the wavelength at which DiOspecifically absorbs. An intense DiI emission at 575 nm was observed,indicating an efficient energy transfer between the closely packed FRETpartners within the nanogel interior (FIG. 7b ). When this experimentwas carried out in acetone, strong DiO fluorescence at 503 nm wasobserved along with significant reduction of DiI emission, suggesting aloss of FRET due to diffusion of the dyes from the nanogel interior tothe solvent. Note that both DiO and DiI are soluble in acetone, butinsoluble in water.

DOPC vesicles can absorb the dye molecules, if available in solution.Therefore, if the dye molecules are not stably encapsulated within thenanogels, the transient presence of these dye molecules in the aqueousmilieu will result in equilibration of the dye between the DOPC vesiclesand the nanogels. This process results in loss of FRET with leakynanogels, due to the sparse distribution of each dye into the bilayer ofDOPC. To investigate the behavior of the nanogels, the fluorescenceintensities of nanogel solutions containing DiO and DiI dyes weremonitored for 3 days by exciting the solution at 450 nm (DiO). Thedisappearance of FRET due to the release of hydrophobic guest moleculeswas then monitored by tracing the increase in the donor (DiO) emissionand the concomitant decrease in the acceptor's (DiI). FIGS. 7c-d showthe change of the dye emission pattern with different crosslinkednanogels, NG1 (6% crosslinked) and NG3 (25% crosslinked). In the case ofthe highly crosslinked nanogel (NG3), the emission of FRET remainedrelatively steady throughout the time of the experiment (FIG. 7d ),indicating that the two dyes are stably trapped inside the nanogels.

Conversely, in the case of the lightly crosslinked nanogel (NG1), theacceptor's (DiI) emission was gradually decreased over the three daysperiod with concurrent increase in the donor's (DiO) emission (FIG. 7c). This result indicates that the hydrophobic guest molecules weretransferred from the nanocontainer to the DOPC vesicle bilayer. Eventhough NG1 showed less encapsulation stability than NG3, the leakage ismuch slower than that from block copolymer micelles previously reported,suggesting the versatility of these nanogels as a delivery vehiclecandidate. (Chen, et al. 2008 Proc. Natl Acad. Sci. U.S.A. 105,6596-6601.) The addition of DTT (20 mM) to the stable nanocontainer(NG3) containing the two dyes led to decreasing FRET (FIG. 7d ). TheFRET ratio I_(a)/(I_(d)+I_(a)) plotted against time, where l_(a) andI_(d) are the maximum emission intensities of the acceptor (DiI) and thedonor (DiO) at 575 nm and 503 nm, respectively, clearly shows thedifference in encapsulation stabilities of these nanogels (FIG. 7e ).This result means that the drug molecules can stably remain inside thenanocontainer during circulation, but be released inside the targetcells in response to the higher reductant (GSH) levels.

To test the in vitro release of guest molecules, nanogels having DiO andDiI co-encapsulated within their interiors were used. In this case, ifthere were no guest release upon cellular internalization, then FRETwould be continually observed within the cytosolic interior. However, ifthe guest release did occur, then the proximity between the DiO and DiIwould greatly increase, causing a qualitative decrease in FRETobservation. The distribution of red (FRET, 585-615 nm spectral filter)and green (no FRET, 505-520 nm spectral filter) fluorescence wasobserved over time by confocal microscopy (λ_(ex)=488 nm). FIG. 8 showsthe fluorescence microscopy images. After 2 h, neither of the nanogelshad gained significant access to the cells (FIGS. 8a and 8d ). Most ofthe red fluorescence was found in the extracellular environment. Theobserved red fluorescence suggests that the dye molecules are stillintact in the polymer nanoparticles. In the case of NG1 (6%crosslinked), green color begins to appear in the cell membrane with alittle red color inside the cell after an incubation period of 4 h (FIG.8b ). The green fluorescence (DiO) in the plasma membrane suggests aninitial loss of FRET due to hydrophobic dye transfer from the nanogelsto the membrane. As time progresses, the extent of fluorescence (bothgreen & red) increases; the observed yellow color is essentially anoverlay of the two colors. After accessing the cell, the greenfluorescence from DiO and the red fluorescence from DiI appear moreequally inside the cells. This indicates that there is a significantrelease of the dye molecules from the nanogel, because there is noenergy transfer that causes the green fluorescence to be suppressed.While the lightly cross-linked gel, NG1, shows initial dye release fromthe nanogels to plasma membrane before cellular internalization, thehighly crosslinked NG3 exhibits complete internalization prior to anysignificant release. This difference is clearly observed in FIG. 8e ,which shows an abundance of red color and a little yellow color insidethe cell with no green color at the membrane after 4 h incubation. At 24h incubation, the intensity of yellow color increased, but the dominantred color indicates slower release due to the dense cross-linking (FIG.8f ). However, after 48 h of incubation yellow color dominates the imageimplying the progression of nanogel disruption and subsequent dyerelease (FIG. 14).

After entry, the less crosslinked nanoparticle NG1 releases the dyemolecules faster than the more crosslinked NG3. It is evident from FIG.8 that the difference between the green and the red fluorescence fromthese cells is much smaller in the case of NG1, compared to NG3. Theseresults indicate that the intracellular GSH acts on NG1 faster than NG3,just as observed with the Nile red release studies outlined above, andare consistent with the dye release differences depending oncrosslinking density from the nanogels to the DOPC bilayer. Importantly,these results demonstrate the tunability in stability of encapsulationand guest release in cells. Also, the cell penetrating processes can beaccelerated using ligands that facilitate internalization by recognizingcell surface receptors. (Ryu, et al. 2010 J. Am. Chem. Soc. 8246-8247.)

Intracellular Delivery of Doxorubicin

In vitro cell viability assays were carried out for the polymernanoparticles prepared above. The nanoparticles are expected to berelatively non-toxic, as they are composed of biocompatible OEG units assurface displays in a methacrylate backbone. Cell viability wasinvestigated by treating 293T human kidney cell lines with nanogels.293T cells were treated with different concentrations of nanogelsolutions and were incubated for 24 h. Cell viability was measured usingthe Alamar Blue assay. As shown in FIG. 9a , the nanogels exhibit highcell viability and no concentration dependent toxicity up to a nanogelconcentration of 1 mg/mL. This result indicates that the nanogelmaterial is non-toxic and thus a potential candidate for biologicalapplications.

To investigate the utilization of the polymer nanoparticles as drugdelivery vehicles, the chemotherapeutic cytotoxic drug molecule,doxorubicin (Dox), was encapsulated during nanogel synthesis by in situloading. The loading capacities were found to be 24 wt % and 20 wt % forNG1 and NG3, respectively. The Dox-loaded nanogels were added to MCF-7cells and the extent of cell death was investigated after 72 h. As shownin FIG. 9b , the Dox-loaded nanoparticles were toxic, but exhibitedslightly lower toxicities than the free drug. This is presumably causedby the delayed release of Dox from the nanogels inside the cells, whilefree Dox molecules easily diffuse through the cellular membrane.Compared to small drug molecules, however, the nanogels are likely to bemore efficient in vivo because of the passive targeting of the nanosizedparticles to tumor tissue by the enhanced permeability and retentioneffect.

Interestingly, both NG1 and NG3 containing Dox showed similar toxicityalthough it was expected that NG1-Dox would be more toxic than NG3-Doxbecause, as described above, the lightly crosslinked NG1 showed fasterdye release in the cell as well as in buffer solution. One possibleexplanation is a difference in the cellular uptake between thenanoparticles of different crosslinking densities. (You, et al. 2009Nano Lett. 9, 4467-4473; Beningo, et al. 2002 J. Cell Sci. 115,849-856.) Differences in uptake were previously observed by in situ dyerelease experiments. As shown in FIGS. 8b and 8e , NG3 exhibited highercellular uptake relative to NG1 at the same time period.

For further study, the cell uptake of both free Dox and Dox-loadednanogels (20 μM Dox concentration) was monitored using confocalmicroscopy over time. Initial internalization of Dox was observed within3 h for free Dox and NG3-Dox, but not until 4 h for NG1-Dox (FIG. 9c-g). While the free Dox showed significant accumulation in the nucleusafter 3 h (FIG. 9c ), NG1-Dox and NG3-Dox only showed strongfluorescence in the cytoplasm (FIG. 9e-g ). Even though NG1 exhibitedfaster dye release in previous experiments, the effective Dox amountreleased from NG3 may be similar to that from NG1 due to the differencesin cellular uptake. This is presumably the reason that the two nanogelsshow similar toxicities.

Pentafluorophenyl Activated Esters and Bifunctional Cross-Linkers

Pentafluorophenyl (PFP) has been used as handles for post-polymerizationmodification with a variety of functional amines. (Eberhardt, et al.2005 Eur. Polym. J. 2005, 41, 1569-1575; Jochum, et al. 2009Macromolecules 42, 5941-5945; Roth, et al. 2008 Macromolecules 41,8513-8519.) While the amidation of PFP is similar to that of theN-hydroxy-succinimide (NHS) activated ester, PFP is relatively morehydrophobic and exhibits high-hydrolytic stability, providingpossibilities of running the reaction in either organic solvent oraqueous solution. (Gibson, et al. 2009 J. Polym. Sci. Part A: Polym Chem47, 4332-4345.) The desired polymer precursors contain poly(ethyleneglycol) as a hydrophilic unit and pentafluorophenyl ester as ahydrophobic unit (FIG. 18b ). The hydrophobic nature of the PFP unitsdrives the polymer precursors to form stable self-aggregates, offeringlipophilic encapsulation capabilities. Additionally, the PFP activatedesters are known to be replaced with amines with great efficiency.Therefore, the addition of diamine cross-linkers to the stable PEG-PFPpolymer aggregates may result in stable cross-linked polymer nanogels,in which lipopohilic molecules can be trapped.

A key feature involves the utility of an amphiphilic random copolymer,where the reactive lipophilic functional groups are utilized forcrosslinking. In this case, pentafluorophenyl moiety is used as thelipophilic functional group that provides such reactivity. Randomcopolymers were prepared containing polyethylene glycol methacrylate(PEGMA) as the hydrophilic unit and the pentafluorophenyl acrylate(PFPA) as the lipophilic unit. Addition of a calculated amount ofdiamine to a solution of the PPFPA-r-PEGMA random copolymer causesinter- and intra-chain crosslinking amidation reactions to afford thenanogel. Since only a percentage of the PFP moiety is used for thecrosslinking reaction, the remaining reactive PFP functionalities can beused for surface functionalization.

Amphiphilic random copolymers (P1′) containing 20% biocompatible, watersoluble poly(ethylene glycol) monomethyl ether (PEG) monomers and 80%hydrophobic pentafluorophenyl activated ester monomers were synthesizedby RAFT polymerization. The resulting polymer was obtained byprecipitation and had a molecular weight of M_(n)=12700 g/mol with anarrow molecular weight distribution (PDI=1.17). Polymer nanogels wereprepared from random copolymer P1′ (20 mg/mL in acetone) by the additionof diamine cross-linkers, including diaminopropane (DAP) and cystamine(CYS or CTM), to initiate cross-linking of the PEG-PFP polymeraggregates. Subsequent addition of water and gradual evaporation of theacetone yielded nanogels NG1′ and NG2′ respectively (FIG. 18b ). Thesizes of the nanogels dispersion in water were observed by DLSmeasurements. At 20 mg/mL, the sizes were observed to be concentrated at110 nm in both water and acetone. Upon dilution of the resultingnanogels to 2 mg/mL in acetone, the sizes retained at 110 nm, while thesizes of the precursor polymer aggregates diluted to the sameconcentration were observed to decrease significantly to around 10 nm.The lack of concentration dependence of the sizes of the nanogels inboth water and acetone suggests that these are stable, cross-linkedparticles. If the formation of these nanogels occurs from preformedpolymer aggregates, the concentration of polymer precursors would affectthe obtained particle size. Indeed, smaller particles (˜10 nm) wereprepared by decreasing the concentration of P1′ to 10 mg/mL during thepreparation reaction.

The amidation of PFP activated esters within the polymer scaffold withdiamine cross-linkers was confirmed by FTIR and ¹⁹F NMR (FIG. 19).Tracing the absorption spectra observed by FTIR reveals that thecarbonyl of PFP ester band clearly shifts from ν=1780 cm⁻¹ to 1660 cm⁻¹over time, indicating amide bond formation from reaction of the PFPester units with the cross-linking functional amines. The additionaladvantage of the using the PFP ester functionality is that analysis ofthe reaction progress can be easily monitored by ¹⁹F NMR spectroscopy asshown in FIG. 19b . The ¹⁹F NMR spectrum of the P1′ precursors showsfour signals at 151.0, 152.6, 159.3, and 164.1 ppm that can be assignedto PFP ester units. After adding the DAP and CYS cross-linkers tocross-link P1′, achieving NG1′ and NG2′ respectively, the evolution ofthree new sharp peaks of the pentafluorophenol leaving groups at 170.4,172.3, and 190.7 ppm indicates progress of the PFP conversion reaction.The remaining PFP activated ester groups provide the possibilities forpost-modification of the nanogels with molecules containing aminefunctionalities. To test this, an excess amount of propylamine was addedto NG1′ and NG2′ to displace the residual PFP units. These post-modifiednanogels were confirmed by FT-IR and ¹⁹F NMR. The disappearance of theabsorption of the carbonyl ester band at 1780 cm⁻¹ and the appearance ofthe carbonyl amide band at 1660 cm⁻¹ suggest that the remaining PFPesters were completely converted to propylamide functional groups. Theprogress of the amidation reaction was also monitored by tracking therelease of the petafluorophenol byproduct. The increase ofpentafluorophenol signals observed by ¹⁹F NMR further confirms thatreplacement of PFP esters with propylamine can be achieved under mildreaction conditions.

To investigate the possibility of encapsulating lipophilic guestmolecules within the formed cross-linked nanogels and to evaluate theencapsulation stability observed in such a carrier scaffold, the FRETbased method employed above was used. In this experiment, the lipophilicFRET pair dyes, DiO (DiOC18(3), FRET donor) and DiI (DiIC18(3), FRETacceptor), were independently loaded into separate nanogel solutions.

Aqueous solutions of NG1′ and NG2′ (2 mg/mL) containing 1 wt % of eitherDiO or DiI were prepared from P1′ (10 mg/mL) stock solution, representedas NGx-DiI and NGx-DiO where x=1′ and 2′. The solutions containing theseparate dyes were then mixed in water (at 10 times dilution).Fluorescence from DiO excitation at 450 nm was monitored over time.Analysis of the linear fit to the first 6 hours of data revealed leakagecoefficients (Λ) of ˜0.009 h⁻¹ and 0 h⁻¹ for NG1′ and NG2′, respectivelysuggesting that the nanogels exhibit high encapsulation stabilities.

The FRET evolution of a NG2′-DiO and NG2′-DiI mixed solution in thepresence of 10 mM GSH was traced. The resulting increase of the FRETratio observed over a 96 hour periods suggests that the encapsulationstability of NG2′ decreases upon disulfide bond cleavage, leading to theleakage or release of the encapsulated dyes molecules (FIG. 20). To testtenability of the encapsulation stability and release rate, NG3′ wasprepared at lower cross-linking densities by decreasing the CTA feedingamount to 0.1 mol % with respect to the P1′ precursors. Dye exchangewith NG3′ in the presence of GSH, as observed by the FRET basedexperiments, is faster than the higher cross-linked NG2′. P1′ aggregatesin water also show high encapsulation stability with Λ=0.001 h⁻¹.However, P1′ aggregates in the presence of 10 mM GSH exhibitdramatically increased of FRET with Λ=0.064 h⁻¹, which is higher thanwhat was observed in the cross-linked nanogels. Note that GSH is atripeptide that contains a primary amine. Thus, GSH can react with PFPunits causing changes in the hydrophobic-lipophilic balance (HLB) of thepolymer aggregates and leading to a loss of encapsulation stability dueto increase solubility of the polymer chains.

In another example, cross-linked nanogels with 50% and 100%cross-linking density (with respect to the PFP units) were prepared froma PPFPA-r-PEGMA solution (10 mg/mL in THF) by adding a calculated amountof CYS and hexamethylenediamine (HMDA) followed by the addition of waterand evaporation of THF. Reaction between the amine the activated esterresults in the formation of the amide bond and the concurrent release ofthe PFP moiety in the form of pentafluorophenol (FIG. 21). IR spectra ofreaction mixtures, listed in FIG. 21a , were recorded after heating at50° C. for 4 h. The peak at 1780 cm⁻¹, corresponding to the activatedester C═O stretching, disappeared with the concurrent appearance of thepeak at 1640 cm⁻¹ ascribed to the amide C═O stretching. When 0.25 eq. ofcross-linker was used, half of the PFP esters were converted to amide.The remaining PFP activated ester C═O signal can still be observed.

The ¹⁹F NMR spectrum of the polymer (FIG. 21b ) shows three broad peaksat −153.4, −159.9, and −164.3 ppm. After adding CYS or HMDAcross-linkers, the polymer was cross-linked to affording nanogels,releasing C₆F₅OH groups, which show sharp signals located at −168.5,−171.4 and −186.6 ppm. The broad peaks fully disappeared with theaddition of 0.5 eq. of cross-linker, suggesting 100% cross-linkingdensity in yielded nanogels. The remaining broad peaks and new sharppeaks are simultaneously observed when 50% of the PFP groups werecross-linked. The dramatic change in chemical shift indicates theconversion from activated ester to amide and hence cross-linking.Additionally, end group analysis by ¹H NMR reveals that most of endgroups remain intact after cross-linking (FIG. 29). The size of thenanogels prepared from a 10 mg/mL polymer solution in THF with CYS weremeasured by DLS shown in FIG. 22.

The size of the nanogel dispersion in water was ˜180 nm, which iscoincident with what was observed from TEM. In order to show that thesynthesized nanogels are stable cross-linked networks, rather than thesimple aggregation of the polymer, nanogels were re-dispersed in THF anda THF/H₂O mixture at various concentrations and were compared. Sizes ofthe same nanogel dispersed in THF or THF/H₂O mixture are similar to thatin H₂O alone. Also, dilution of the nanogels to 2 mg/mL in THF or H₂Odoes not result in a size change (remains at ˜180 nm). However, the samedilution of the polymer aggregates, prior to the crosslinking reaction,reduces the aggregate sizes to ˜13 nm. These results suggest that thenanogel is from a covalently crosslinked network and has noconcentration and solvent dependency. A similar result was observed fromthe nanogel cross-linked by HMDA (FIG. 27). Although PPFPA-r-PEGMAdissolved in THF shows peaks at around 10 nm, the poor correlationfunction is taken to indicate ill-defined aggregation. Since the nanogelsizes are much larger than the aggregate sizes of PPFPA-r-PEGMA in THF,there is some inter-aggregate crosslinking under these reactionconditions.

A lipophilic guest molecule, DiI was encapsulated in nanogels duringcross-linking. During this encapsulation, 1 wt % of DiI with respect topolymer was initially fed. Guest molecule encapsulation was measured byabsorbance spectroscopy shown in FIG. 23.

Nanoparticle size is known to play a significant role in severalapplications. For example, in the case of drug delivery, size has impacton biodistribution, cellular uptake, and permeability in disease sites,thus affecting the therapeutic efficacy. (He, et al. 2010 Biomaterials,31, 3657; Balogh, et al. 2007 Nanomed-Nanotechnol, 3, 281; Wong, et al.2011 Natl Acad Sci USA, 108, 2426.) Larger nanogel size is a result ofsmall percentage of inter-aggregate cross-linking in addition to thedesired intra-aggregate crosslinking. The inter-aggregate phenomenon isexpected to be highly concentration dependent. Hence, the nanogel sizewould be significantly affected by the original concentration of thePPFPA-r-PEGMA solution. Four nanogels were prepared from 2 mg/mL, 5mg/mL, 10 mg/mL and 20 mg/mL PPFPA-PEGMA solution in THF. As shown inFIG. 24, the size of the nanogel dispersed in H₂O shifts from 100 nm to200 nm along with the increase in PPFPA-PEGMA concentration.

Preparing the nanogels directly in water offers several advantages: (i)the organic solvent free nanocarrier preparation method is muchpreferred in terms of removing the trace amounts of solvent and itsenvironment-friendly nature; (ii) allows for an additional handle totune the particle size; (iii) allows for efficient incorporation of abroader range of lipophilic guest molecules within the nanogels.(Nilles, et al. 2011 Polym. Chem. 2, 376; Gibson, et al 2009 J. Polym.Sci. Pol. Chem. 47, 4332.)

Nanogels were prepared in aqueous solutions using a PPFPA-r-PEGMAsolution at a variety of polymer concentrations. The cross-linkingreaction in H₂O was also detected by FTIR. The collected FTIR spectrumof the aqueous reaction mixture after 4 h heating was shown in FIG. 25a. No remaining PFP groups were observed after cross-linking, indicatedby the complete disappearance of the activated ester C═O absorbance.Evolution of a peak at 1640 cm⁻¹ further confirms the amidation. Anotherpotential issue is the extent to which the PFP activated ester wasconverted to amide, compared to the potential hydrolysis of the esterduring the cross-linking process. This can be addressed by monitoringthe IR spectrum. The absorbance of the poly methacrylic acid, theproduct of hydrolysis, should be observed if hydrolysis occurred duringthe cross-linking process. However, no significant evidence was observedfor the hydrolysis peak, which is expected to be at around 1710 cm⁻¹.(Billingham, et al. 1997 Vibrational Spectroscopy, 14, 19; Arndt, et al.1999 Acta Polym. 50, 383; Dubinsky, et al. 2005 J. Polym. Sci. Polym.Phys. 43, 1168.) This suggests that cross-linking is the predominantreaction. Interestingly, the sizes of the nanogels prepared in H₂O werearound 10 nm independent of the concentration of PPFPA-PEGMA solutionsused. Size measurements from DLS are in good agreement with the TEMresults shown in FIG. 25 b.

The residual PFP moieties can be utilized to additionally functionalizethe surface of the nanogels, providing at least two key advantages: (i)this post-nanogel formation reaction can be used to eliminate theremaining reactive PFP moieties. This is useful in applications such asdrug delivery, where the reactivity of the PFP moiety could be a sourceof toxicity; (ii) this allows for the incorporation of functionalitieson the surface of the nanogels. An example of an implication of such acapability includes incorporation of ligands for sensing and targeteddelivery.

In order to demonstrate the possibility of surface engineering on thesenanogels, isopropylamine (IPA) and N,N-dimethylethylenediamine (DMEDA)were incorporated onto the 50% crosslinked nanogel after the nanogelsynthesis. This reaction with IPA and DMEDA was monitored by FTIR (FIG.28), which clearly indicates that the amidation step can indeed becarried out in the post nanogel synthesis steps. Moreover, zetapotentials of these nanogels were measured to obtain additional evidencefor surface modification (FIG. 26). Zeta potentials of the 50% and 100%cystamine crosslinked nanogels were around −30 mV. Similarly, the 50%cross-linked nanogel modified by IPA also has a zeta potential of −30mV. The observation of negatively charged nanogel surface is surprising.However, when the nanogel was modified by DMEDA, the zeta potential wasfound to shift to +5 mV. The positively charged surface is attributed tothe protonation of DMEDA and therefore taken to designate surfacemodification. The presence of these functionalities on the nanogelsurface can also be presumed, because prior studies with the disulfidecrosslinked nanogels show that cell penetrating peptide ligands areindeed available on the suface after a similar functionalization step.(Ryu, et al. 2010 J. Am. Chem. Soc. 132, 8246.)

Thus, the invention provides a facile methodology to achieve polymericnanogels using a simple reaction between the lipophilic activated PFPester and diamines. This strategy has an advantage that the syntheses ofnanogels are not limited to disulfide crosslinked systems orself-crosslinking reactions. The intermolecular nature of thecrosslinking reaction allows for incorporating a broader variety ofstimuli-sensitive features in the diamine crosslinkers and thus in thenanogel. Advantages of the disclosed approach include: (i) there issignificant tunability in the size of the nanogels, especially in THF;(ii) these nanogels can encapsulate lipophilic guest molecules duringthe crosslinking step of the nanogel synthesis; (iii) the nanogels aredispersible in water, irrespective of whether they were prepared in THFor water; (iv) residual PFP moiety can be used in a post-nanogelassembly step to incorporate surface functionalities.

Concurrent Binding and Delivery of Proteins and Lipophilic SmallMolecules

Nanoscale vehicles are desired that can concurrently sequester anddeliver two different molecules. (Kelkar, et al. 2011 Bioconjug. Chem.22, 1879-1903; Kelkar, et al. 2011 Acc. Chem. Res. 44, Issue #10; Jain2001 J Nature Med. 7, 987-989; Sengupta, et al. 2005 Nature, 436,568-572.) It is less challenging when two lipophilic or two hydrophilicmolecules are co-sequestered and delivered. It is much more difficultand complicated when a combination of a water-soluble hydrophilicmolecule and a water-insoluble lipophilic molecule are to beco-encapsulated and delivered, especially so when one of the cargos is aprotein. The propensity of proteins to irreversibly denature undernon-native conditions presents a significant challenge. (Kim, et al.2009 Langmuir 25, 14086-14092; Kim, et al. 2011 Mol. Pharmaceutics. 8,1955-1961; Wiradharma, et al. 2009 Biomaterials 30, 3100-3109.)

As schematically illustrated in FIG. 30, the invention utilizes thehydrophobic interior of a polymer assembly to stably encapsulatelipophilic guest molecules, while utilizing the complementaryelectrostatic interaction between the surface of the polymer assemblywith a peptide to bind proteins. β-galactosidase (β-gal, pI: 4.8, fromE. coli) is used as the model protein for testing, which offers thebenefit of studying the activity of this enzyme both inside and outsidethe cells. DiI dye is used as the model small molecule, which islipophilic and exhibits fluorescence characteristics (complementary tothe fluorescein labeling of the protein) allowing for concurrentmonitoring of the protein and the small molecule in solution and insidethe cells. Self-crosslinked nanogels of the invention are used as thenanocarrier, which offer the capabilities for stably encapsulatinglipophilic dye molecules. (Ryu, et al. 2010 J. Am. Chem. Soc. 132,17227-17235; Jiwpanich, et al. 2010 J. Am. Chem. Soc. 132, 10683-10685.)

Two nanogels, NG1 and NG2, were prepared with 6% and 14% crosslinkingdensities respectively. As provided herein, the nanogels were preparedfrom a random copolymer, obtained using a hydrophilicoligoethyleneglycol methacrylate and a lipophilic methacrylate monomercontaining a PDS moiety. The self-assembled structure was “locked in”with the non-covalently sequestered lipophilic guest DiI molecules byinitiating a thiol-disulfide exchange reaction among the PDS units usingDTT. (Jiwpanich, et al. 2010 J. Am. Chem. Soc. 132, 10683-10685.) Theextent of crosslinking was controlled by the amount of DTT added to thereaction mixture.

The surface of this nanogel contained charge-neutral functional groups.Positively charged functional groups, tri-arginine, were introduced tobind to the negatively charged surface of β-gal. This tripeptide canperform the dual role of providing the positive charge and alsoexhibiting cell-penetrating peptide characteristics. (Rothbard, et al.2004 J. Am. Chem. Soc. 126, 9506-9507; Nakase, et al. 2008 Adv. DrugDeliv. Rev. 60, 598-607.) Since only a small percentage of the PDSgroups were used for the crosslinking reaction, the residual PDSmoieties conjugated the tri-arginine moiety using a Cys-Arg-Arg-Arg(CRRR) peptide. Here, the thiol moiety in cysteine reacts with the PDSunits to provide the targeted nanogel. The structures of the nanogel'spolymer precursor and the CRRR peptide are shown in FIG. 31.

In the nanogel formation process, NG1 showed only a small amount ofproduction of pyridiothione (the byproduct of disulfide crosslinking) at330-340 nm. On the other hand, NG2 showed a large amount ofpyridiothione release indicating that NG2 is more densely crosslinked(FIG. 36). Subsequent CRRR addition showed a further increase in theabsorption peak of pyridiothione, indicative of peptide conjugation ontothe surface of the nanogels. The same amount of CRRR addition showed asmaller increase for NG2 compared to NG1. This means that the degree ofmodification with CRRR peptide is much more for NG1 than for NG2. Thisis because the remaining PDS groups for surface modification afternanogel formation are less at high crosslinking densities. Thus, the twonanogels have different degrees of surface functionalization, whichshould have implications in protein binding and delivery. The absorptionintensity of hydrophobic dye, DiI, did not change during the surfacemodification step, indicating that peptide modification process doesn'taffect the encapsulation stability of the hydrophobic guest in thenanogel.

Next, β-gal functionalized with fluorescein (FITC-β-gal) was used totest binding of the nanogels. The absorption and emission spectra of DiIand fluorescein suggest that these two dye molecules can be ideal FRETpartners. Using FRET, it was found that the optimum ratios of NG1-CRRRand NG2-CRRR complexation with β-gal were 2:1 and 4:1, respectively.NG1-CRRR showed stable complexation at a lower concentration thanNG2-CRRR, indicating stronger binding by the former nanogel, likely dueto the greater amount of the peptide on the NG1-CRRR surface.

Also evaluated were the size change and charge alteration of the nanogelsurface due to the complexation event using DLS and zeta potentialmeasurements respectively. The hydrodynamic diameters of individualNG1-CRRR, NG2-CRRR, and β-gal were found to be approximately 20 nm, 12nm, and 10 nm, respectively (FIGS. 32a and 32b ). Once the nanogels andthe protein were mixed, the size shifts to about 40 nm for the complexof NG1-CRRR/β-gal and 30 nm for NG2-CRRR/β-gal complex. Based on thesizes of the nanogels, β-gal, and the complexes, combined with theirratio, the average numbers of nanogel and β-gal per complex wereestimated to be 7.6 and 3.8, respectively, for NG1-CRRR and 13.6 and 3.4for NG2-CRRR. Zeta-potential studies showed that the apparent surfacecharge of the nanoparticles shifted towards the negative values, from+25 mV to −5 mV for NG1-CRRR and from 0 mV to −20 mV for NG2-CRRR afterβ-gal incorporation (FIGS. 32c and 32d ). Nanogel-protein complexationwas also reinforced by gel electrophoresis studies (FIG. 38). While,NG2-CRRR showed a large amount of unbound β-gal, NG1-CRRR showed anegligible amount of free β-gal. These results demonstrated that whileboth nanogels bind β-gal, the more positively charged NG1-CRRR exhibitsstronger binding to the protein.

The key motivation in developing this system is to achieve efficientintracellular delivery of the hydrophilic protein along with thelipophilic small molecules. It is important to maintain stability andactivity of the delivered protein throughout the process. (Rothbard, etal. 2004 J. Am. Chem. Soc. 126, 9506-9507; Nakase, et al. 2008 Adv. DrugDeliv. Rev. 60, 598-607.) To this end, the activity of the protein wastested when complexed to the nanogel in solution before performing anyintracellular release studies. An enzyme activity assay was done forβ-gal complexed to NG1-CRRR and NG2-CRRR after 1 h incubation with thenanogels. It was observed that the protein retains its activity evenwhen bound to the surface of the nanogels (FIG. 39). These resultsdemonstrate the robustness of the complex in maintaining proteinactivity.

Intracellular delivery of DiI and β-gal was also tested. As the nanogelsconsist of biodegradable disulfide crosslinkers, the release of thecomplexed protein and encapsulated lipophilic molecule can be triggeredupon exposure to glutathione (present at higher concentrations insidethe cells (mM), compared to the extracellular concentration (μM)). Tocompare the release profile of these dual delivery systems, NG1-CRRR andNG2-CRRR containing DiI in its interior and complexed with FITC labeledβ-gal, were added to HeLa cells with serum and without serum. In thecase of cell internalization of both encapsulated DiI and FITC labeledprotein, a yellow color would be observed due to the co-localization andoverlay of both red and green fluorophores for DiI and FITC,respectively. If there is no internalization of the nanogel, no colorcould be observed.

HeLa cells were treated with a 2:1 ratio of NG1-CRR/FITC-β-gal complexesand a 4:1 ratio of NG2-CRRR/FITC-β-gal complexes. Fluorescencedistribution of DiI and FITC was observed over time by confocalfluorescence microscopy with simultaneous excitation of both dyemolecules at 488 nm and 543 nm for FITC and DiI, respectively. As shownin FIGS. 33a and 33b , green and red emissions were observed when thecells were treated with the nanogel-protein complexes. On the otherhand, cells incubated with FITC-β-gal alone displayed no fluorescence,indicating that the protein is not capable of cell penetration by itself(FIG. 40). The green fluorescence of the FITC (label for protein) andred fluorescence for DiI (encapsulated small molecule) appear uniformlydistributed. While co-localization of DiI dye and β-gal in cells bothwith and without serum was observed for NG1-CRRR (FIGS. 33a and 33c ),NG2-CRRR showed almost no efficiency of intracellular delivery of eithercargos in the presence of serum (FIGS. 33b and 33d ). The differences indelivery efficiency appear to be due to the stronger interaction of theprotein with NG1-CRRR compared to NG2-CRRR, where serum proteins appearto compete for the interaction with the nanogels. The presence of redfluorescence with the yellow fluorescence in the case of the NG1-CRRRcomplex indicates that the nanogel and the protein are not onlyinternalized, but also are also starting to spatially separate from eachother, presumably due to GSH-based degradation of the disulfide bonds.

X-gal assays were performed on cells, which had been incubated with thenanogel/β-gal complex, to probe the intracellular activity of thedelivered β-gal (FIG. 33e ). The observed blue color is due to theconversion of the 5-bromo-3-indoyl-β-D-galactopyranoside substrate to anintensely blue colored indigo derivative, by β-gal. From the opticalmicroscope images, it can be observed that the cells treated withprotein conjugated NG1-CRRR show enhanced blue coloration, due togreater β-gal internalization compared to NG2-CRRR and the controlcells. These studies demonstrate that the delivered β-gal is activeinside the cells. The observation that protein bound to NG1-CRRR hasbetter intracellular activity than NG2-CRRR is consistent with theinternalization studies using confocal microscopy. The results hereinshow that concurrent delivery of a protein and a hydrophobic dyesimultaneously is indeed feasible with the approach outlined here.

Thus, the invention provides a nanogel that is capable of encapsulatinglipophilic small molecules within its crosslinked interiors and bindingproteins on its surface through electrostatic interactions. The nanogelscan be functionalized with cell penetrating peptides efficiently. Thenanogels bind oppositely charged proteins and that the charge density onthe nanogel surface affects the efficiency of binding of thecomplementarily charged proteins. Complexation of the protein with thenanogel does not alter the activity of the protein. The complex exhibitsefficient uptake by cells, where both the lipophilic small molecule andthe protein are concurrently taken up by the cells. The enzyme retainsits activity even upon cellular entry. The design strategy outlined herecould have broad implications in a variety of areas includingtherapeutics, diagnostics, and a combination of the two by way ofnanotheranostics.

Ligand-Decorated Nanogels and Cellular Targeting

Ligand-modified nanocarriers that are capable of targeting specificcells hold great promise in therapeutic applications, such as cancerchemotherapy. (Brannon-Peppas, et al. 2004 Adv. Drug Delivery Rev. 56,1649-1659; Davis, et al. 2008 Nat. Rev. Drug Discovery 7, 771-782;Kabanov, et al. 2009 Angew. Chem. Int. Ed. 48, 5418-5429; Duncan 2003Nat. Rev. Drug Discovery 2, 347-360; Peer, et al. 2007 Nat. Nanotechnol.2, 751-760.) When such nanoscale platforms also exhibit the propensityto act as carriers for non-covalently loaded cargo, these implicationsare even greater.

Targeting mechanisms for nanocarriers in cancer chemotherapy can bebroadly classified into two categories, active and passive. Passivetargeting is based on the propensity of nanoscopic objects, with 10-200nm sizes, to selectively accumulate in solid tumor tissue due to theincreased permeability of the tumor vasculature and ineffectivelymphatic drainage, which is known as the enhanced permeation retention(EPR) effect. (Baban, et al. 1998 Adv. Drug Delivery Rev. 34, 109-119;Maeda, et al. 2000 J. Controlled Release, 65, 271-284.) Active targetingis achieved by associating the nanocarrier with ligands that exhibithigh affinity for receptors that are overexpressed on the cancer cellsurface. (Allen 2002 J. Controlled Release 2, 750-763; Byrne, et al.2008 Adv. Drug Delivery Rev., 60, 1615-1626.) While either of thesetargeting approaches can be effective, the most successful approach willlikely be achieved by combining the key features of both strategies.

Targeting ligands, such as folic acid, have been studied as componentsof active targeting systems in the field of drug delivery. (Nasongkla,et al. 2004 Angew. Chem. Int. Ed. 43, 6323-6327; Lee, et al. 2008 Angew.Chem. Int. Ed., 47, 2418-2421; Aluri, et al. 2009 Adv. Drug DeliveryRev., 61, 940-952; Sudimack, et al. 2000 Adv. Drug Delivery. Rev. 41,147-161.) In many cases, these ligands are attached to the hydrophilicends of assembly-forming amphiphilic molecules (e.g. block copolymers).(Sutton, et al. 2007 Pharm. Res. 24, 1029-1046; Yoo, et al. 2004 J.Controlled Release 96, 273-283; Xu, et al. 2007 Angew. Chem. Int. Ed.46, 4999-5002.)

However, the installation of such ligands onto these molecules requirescomplicated synthetic steps, limiting the versatility of ligandfunctionalization to tailor carriers for targeting specific cell types.Additionally, modification of the self-assembling molecules by thismethod could alter their hydrophilic-lipophilic balance (HLB) andintroduce undesirable variations in assembly behavior, thus requiringoptimization on a case-by-case basis.

A class of drug delivery vehicle, which affords surface modification ofchemically-crosslinked nanocarriers with reactive functional groups in apost-assembly step, can address these complications. In such a system,the nanocarrier morphology is maintained during modification, providingversatility in surface functionalization without compromising thestructural integrity of assembly properties of the nanoscopic scaffold.The nanocarrier hosts stably encapsulate guest/drug molecules until theyreach the site of the targeted cells.

Herein disclosed is a fast, one-pot synthesis of ligand-decoratednanogels (FIG. 41). The resulting delivery vehicles exhibit highencapsulation stability to exploit passive targeting mechanisms and canbe easily modified at their surfaces with various ligands for the activetargeting of cell surface receptors.

The targeted nanogels (T-NGs) were prepared within 2 h by a one-potsynthesis and exhibited very narrow size distributions. These nanogelscan be simply prepared with cysteine-modified ligands including folicacid, cyclic arginine-glycine-aspartic acid (RGD) peptide, andcell-penetrating peptide. As demonstrated herein, the T-NGs hold theirpayloads, undergo facilitated cell internalization by receptor-mediateduptake, and release their drug content inside cells due to cleavage ofcrosslinked disulfide bonds by the reducing intracellular environment.(Vader, et al. 2011 Pharm. Res. 28, 1013-1022; Lin, et al. 2009 J.Expert Opin. Drug Deliv. 6, 421-439; Bronich, et al. 2002 J. Am. Chem.Soc. 124, 118720-11873; Kim, et al. 2010 Biomacromolecules 11, 919-926.)

Preparation of T-NG. T-NGs were prepared through the lock-in strategyusing a random copolymer that contains oligoethylene glycol (OEG) units(29%) and PDS moieties (71%) as side chain functionalities (FIG. 42). Atelevated temperatures, the polymer formed larger aggregates (42 nm indiameter) than at room temperature (24 nm in diameter), likely due tointermolecular associations between the polymer chains caused by LCSTbehavior of the OEG units (FIG. 43a ). Preparation at high temperaturesfacilitates the crosslinking reaction and that subsequent addition ofcysteine-containing ligands would enable a fast, one-pot T-NG synthesis.The crosslinking and surface modification reactions were observed tofinish within a very short time period. FIG. 43b , which tracesproduction of the pyridothione byproduct of disulfide bond formationduring nanogel synthesis, plateaus within 20 min. A deficient amount ofDTT (20 mol % against the precursor PDS groups) cleaved a correspondingamount of polymer PDS functionalities, generating free thiols that canthen react with remaining PDS functionalities in the polymer chain (bothintra- and inter-chain) to provide the crosslinked polymer nanogel.Based on pyridothione release, the actual crosslinking density was foundto be 13-14% (39 mol % of PDS is consumed), which is very close to thetheoretically calculated crosslinking density of 14%.

The cysteine-containing ligand, cysteine-triarginine (CRRR) peptide, wasadded to this reaction mixture to modify the surface and attachment ofthe ligand was evaluated by further increase in pyridothione absorption.The peak became saturated within 60 min and it was calculated that 17mol % of total PDS is reacted with CRRR (FIG. 43c ). It was estimatedthat there were about seven thousand ligands per T-NG. One can questionwhether such a thiol-containing ligand will cleave the crosslinkingdisulfide bonds, resulting in size change, nanogel disassembly, andleakage of hydrophobic guest molecules during ligand modification. T-NGretained their size from the polymer aggregate at high temperature (FIG.43a ), and the absorption intensity of hydrophobic dye, DiO, which isencapsulated prior to crosslinking and surface modification was notchanged, indicating that the nanogels were sufficiently stable to retaintheir guest molecules during this functionalization period (FIG. 43c ).Surface modification was confirmed of the nanogels by monitoring thechange of surface charge. As shown in FIG. 43d , the nanogels modifiedwith CRRR (NG-RRR) showed positive zeta potentials (+4 mV), while thenanogels showed negative zeta potentials (−20 mV) before surfacemodification.

The resulting T-NGs showed narrow size distribution. Atomic forcemicroscope (AFM) images reveal well-defined spherical structures withvery uniform size. The size is around 50 nm in diameter, which may beideal for the passive targeting applications. (Chithrani, et al. 2007Nano Lett. 7, 1542-1550.) Additional ligands, includingcysteine-modified folic acid and cysteine-containing cyclicarginine-glycine-aspartic acid (RGD) peptides, were also linked by thissynthetic method to make T-NGs, NG-FA and NG-RGD, respectively.

Several features are noteworthy in the modified synthetic methods: (i)the current method allows for obtaining these nanogels in very shortamount of time; (ii) it also allows for decorating the nanogels withtargeting ligands in a single pot and the precursor polymer iscrosslinked into a nanogel and decorated with targeting ligands in lessthan two hours; (iii) the resultant nanogels exhibit narrowpolydispersity; and (iv) these nanogels also exhibit enhancedencapsulation stability. (Ryu, et al. 2010 J. Am. Chem. Soc. 132,8246-8247; Ryu, et al. 2010 J. Am. Chem. Soc. 132, 17227-17235.)

Encapsulation stability. The noncovalent encapsulation stabilities ofthe T-NGs were tested from the dynamics of guest interchange in thenanocarriers using a FRET experiment. (Jiwpanich, et al. 2010 J. Am.Chem. Soc. 132, 10683-10685.) Two lipophilic FRET pair dye molecules,DiO and DiI, were independently encapsulated in the nanogels. Whilesignificant and rapid FRET evolution was observed in the case of thepolymer aggregates, the crosslinked nanogels showed no significant peakshift, indicating their high encapsulation stabilities (FIG. 44).

Cellular internalization of polymer micelle and NG. The differences incellular internalization of the polymer aggregates, nanogels and T-NGsloaded with hydrophobic dye molecules were tested. The polymeraggregates and nanogels, containing DiO as a hydrophobic dye, were addedinto three different cell cultures (293T, MCF-7, and HeLa cell lines).The uptake was monitored by tracing the dye's fluorescence usingconfocal microscopy (FIG. 45a-b ). In the case of the polymeraggregates, intense green emission inside the cells was observed within6 h across all cell lines. In contrast, the nanogels showed no emission,indicating that the nanogels were not internalized in this short timeperiod. Also performed were in situ FRET experiments with HeLa cellsusing the amphiphilic aggregates in which the FRET pair DiO (greenemission) and DiI (red emission) was co-encapsulated. In this case, ifthe polymeric aggregates were internalized through the membrane, thenFRET (red emission, 585-615 nm spectral filter) would be continuallyobserved in the cytosolic interior within a short time period. After 3 hincubation, it was observed that green (no FRET, 505-520 nm spectralfilter) fluorescence is dominant inside the cell by confocal microscopy(ex=488 nm), indicating that the two dyes are not close together withinthe assembly core, but that they have released from the micelles (FIG.45c ). This demonstrates that liphophilic guests loaded inside micellaraggregates can be transferred into any cells, resulting in non-specificdelivery. High encapsulation stability is essential to preventpremature, non-triggered release and achieve selective delivery of thedrug molecules to target cells.

Selective cellular internalization of T-NG. To promote selective andrapid internalization into target cells, the surfaces of theslow-internalizing nanogels were decorated with specific ligands. Toprobe the versatility of ligand functionalization, three differentligands were used: CRRR, CRGD, and cysteine modified-folic acid. RGD isthe ligand of the integrin α_(v)β₃ that is involved in tumorangiogenesis and metastasis and is overexpressed in many solid tumortypes such as breast, prostate, and ovarian cancers. (Haubner, et al.1996 J. Am. Chem. Soc. 118, 7461-7472.) Folic acid is a widely usedligand in targeted delivery research, because folate receptors (FR) arenotably overexpressed on the surfaces of many cancer cells such asovarian, lung, and uterine tumors. (Lu, et al. 2002 Adv. Drug DeliveryRev. 54, 675-693.) RRR can be considered as a positive control, as it isa model cell penetrating peptide and is thus rapidly and non-selectivelytaken up by most cell types. These arginine-rich peptides are consideredto be mimics of the well-known TAT peptides, which are known fortranslocating molecules and nanoscale objects across the cellularmembrane. (Rothbard, et al. 2005 Adv. Drug Delivery Rev. 57, 495-504.)

Cellular uptake of the nanogels modified with CRRR, CRGD, andcysteine-modified-folic acid (referred to as NG-RRR, NG-RGD, and NG-FA,respectively) were tested in varied cell lines: 293T kidney cells arenormal cells used as a control, MCF7 is a slightly FR-positive tumorcell line and is negative to RGD, while SKOV3 and HeLa cells arepositive to both ligands.

To test selective cellular internalization by receptor-mediatedendocytosis, ligand-decorated nanogels encapsulating DiO as a modelhydrophobic drug molecule were incubated with various cell lines for 6 hand then qualitatively examined through confocal microscopy (FIG. 46).In the case of NG-RRR, high green emission was observed inside cells inall cell lines within just 2 h, indicating that the nanogels wereinternalized rapidly because of the highly positive cell penetratingpeptide, RRR. In the case of NG-FA, the nanogels exhibited fast cellularuptake with SKOV3, HeLa and MCF7 cells in which FR is overexpressed.However, the FR-negative 293T cells showed negligible cellular uptake ofthe folic acid decorated nanogel. Similarly, with NG-RGD, HeLa and SKOV3exhibited high internalization, as these cells are considered tooverexpress integrin α_(v)β₃. However, MCF7 and 293T cells, which arenegative to integrin, showed little internalization. These resultsdemonstrate the selective internalization of the various T-NGs throughreceptor mediated endocytotic pathways.

Selective delivery of chemotherapeutic drug. To test whether theobserved target selectivity would translate into differential cell killefficiency, the ligand-coated nanogels encapsulating a chemotherapeuticdrug molecule, paclitaxel (PTX) were prepared. The PTX-loaded polymericaggregates (polymer-PTX), NG (NG-PTX) and NG-RGD (NG-RGD-PTX) were addedto MCF7 cells and HeLa cells and the extent of cell death wasinvestigated after 72 h. As shown in FIG. 47, free PTX and polymer-PTXwere highly toxic in both cell lines. This supports the hypothesis thatthe polymeric micellar aggregates have a leaky nature, which leads tofree drug being translocated through the cellular membrane. In contrast,NG-PTX showed less toxicity, compared to both free-PTX and polymer-PTX.This is consistent with the observed slow internalization of nanogels,which should result in a reduced amount of chemotherapeutic inside thecells. Remarkably, NG-RGD-PTX showed different cell killing efficiencyin two different cell lines. While MCF7 showed similar toxicity withNG-PTX, HeLa showed high toxicity that is similar to free-PTX,indicative of the selective delivery of PTX to this cell line. Togetherwith the cellular internalization experiments, this result demonstratesthat NG-RGD is highly internalized into the integrin receptoroverexpressing HeLa cells, thus enhancing the efficiency ofchemotherapeutics by promoting selective drug delivery.

Thus, the invention provides a facile synthetic method for thepreparation of nanogels: (i) these nanogels can be prepared in under twohours from their polymeric precursor; the reaction time includes theligand decoration step; (ii) these nanogels exhibit high encapsulationstability of lipophilic guest molecules; (iii) the facile ligandfunctionalization possibility can be utilized to decorate these nanogelswith cell targeting ligands; (iv) while the unfunctionalized nanogelsare taken up very poorly by various cells, the ligand-decorated nanogelsexhibit facilitated receptor-dependent cellular uptake, as demonstratedby selective uptake of RGD- and folic acid-decorated nanogels by cellsoverexpressing integrin and folate receptors; (v) functionalization ofthe nanogels with cell penetrating peptides caused rapid non-specificuptake by the cells, independent of the receptor; (vi) the selectiveinternalization capability can be translated to delivering achemotherapeutic drug molecule specifically to a specific receptor-richcell. Overall, the reported versatile one-pot synthetic method forsynthesizing the ligand-decorated nanogels, combined with the intrinsicencapsulation stability and targeting capabilities of the formed T-NGs,should open up new avenues in targeted drug delivery for crosslinkedpolymer nanogels.

EXPERIMENTAL

General. 2,2′-Dithiodipyridine, 2-mercaptoethanol, polyethylene glycolmonomethyl ether methacrylate (MW 450), D,L-dithiothreitol (DTT),1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), Nile red, Tat-SH,and other conventional reagents were obtained from commercial sourcesand were used as received unless otherwise mentioned. Polymer wassynthesized using RAFT polymerization and then purified byprecipitation. S-dodecyl-S′-2-(2,2-dimethylacetic acid)trithiocarbonate, (Lai, et al. 2002 Macromolecules 35, 6754-6756.)2-Cyano-2-propyl benzodithioate and pyridyl disulfide ethyl methacrylate(PDSEMA) was prepared using a previously reported procedure. (Brown, etal. 2007 Chem. Commun. 2145-2147; Ghosh, et al. 2006 Macromolecules 39,5595-5597.) ¹H-NMR spectra were recorded on a 400 MHz Bruker NMRspectrometer using the residual proton resonance of the solvent as theinternal standard. Molecular weights of the polymers were estimated bygel permeation chromatography (GPC) using PMMA standard with arefractive index detector. DLS measurements were performed using aMalvern Nanozetasizer. The fluorescence spectra were obtained from aJASCO FP-6500 spectrofluorimeter. Transmission electron microscopy (TEM)images were taken from JEOL 100CX at 100 KV.

Random copolymer 1. A mixture of 2-Cyano-2-propyl benzodithioate (3 mg,0.0135 mmol), PDSEMA (52 mg, 0.203 mmol), polyethylene glycol monomethylether methacrylate (96.7 mg, 0.203 mmol) and AIBN (0.11 mg, 0.6 μmol)was dissolved in THF (300 μL) and N₂ gas was purged 30 min to thesolution. To this reaction mixture, 500 μL THF was added and sealed andthen put into a pre-heated oil bath at 62° C. for 40 h. To removeunreactive monomers, the resultant mixture was precipitated in coldethyl ether (20 mL) to yield the random copolymer as a waxy liquid. GPC(THF) M_(n): 6.9 K. PDI: 1.2. ¹H NMR (400 MHz, CDCl₃) δ: 8.45, 7.66,7.09, 4.20-4.06, 3.90-3.36, 3.01, 2.15-1.62, 1.43-0.86. The molar ratiobetween two blocks was determined by integrating the methoxy proton inthe polyethylene glycol unit and the aromatic proton in the pyridine andfound to be 4.7:5.3 (PEO:PDSEMA).

Random copolymer 2. A mixture of 2-Cyano-2-propyl benzodithioate (3 mg,0.0135 mmol), PDSEMA (104 mg, 0.407 mmol), polyethylene glycolmonomethyl ether methacrylate (193.4 mg, 0.407 mmol) and AIBN (0.22 mg,1.3 μmol) was dissolved in THF (300 μL) and N₂ gas was purged 30 minutesto the solution. To this reaction mixture, 500 μL THF was added andsealed and then put into a pre-heated oil bath at 62° C. for 40 h. Toremove unreactive monomers, the resultant mixture was precipitated incold ethyl ether (20 mL) to yield the random copolymer as a waxy liquid.GPC (THF) M_(n): 13 K. PDI: 1.2. ¹H NMR (400 MHz, CDCl₃) δ: 8.46, 7.68,7.11, 4.35-4.09, 3.94-3.37, 3.03, 2.04-1.64, 1.43-0.87. The molar ratiobetween two blocks was determined by integrating the methoxy proton inthe polyethylene glycol unit and the aromatic proton in the pyridine andfound to be 5.0:5.0 (PEO:PDSEMA).

Random copolymer 3. A mixture of 2-Cyano-2-propyl benzodithioate (3 mg,0.0135 mmol), PDSEMA (77.6 mg, 0.304 mmol), polyethylene glycolmonomethyl ether methacrylate (61.89 mg, 0.130 mmol) and AIBN (0.22 mg,1.3 μmol) was dissolved in THF (300 μlL and N₂ gas was purged 30 min tothe solution. To this reaction mixture, 500 μL THF was added and sealedand then put into a pre-heated oil bath at 62° C. for 40 h. To removeunreactive monomers, the resultant mixture was precipitated in coldethyl ether (20 mL) to yield the random copolymer as a waxy liquid. GPC(THF) M_(n): 14.4 K. PDI: 1.6. ¹H NMR (400 MHz, CDCl₃) δ: 8.44, 7.65,7.08, 4.29-4.05, 3.78-3.35, 3.00, 2.02-1.66, 1.34-0.87. The molar ratiobetween two blocks was determined by integrating the methoxy proton inthe polyethylene glycol unit and the aromatic proton in the pyridine andfound to be 3.3:6.7 (PEO:PDSEMA).

Random copolymer 4. A mixture of S-dodecyl-S′-2-(2,2-dimethylaceticacid) trithiocarbonate (90 mg, 0.28 mmol), PDSEMA (5 g, 19.6 mmol),polyethylene glycol monomethyl ether methacrylate (4 g, 8.4 mmol) andAIBN (10 mg, 0.056 mmol) was dissolved in DMF (10 mL) and degassed byperforming three freeze-pump-thaw cycles. The reaction mixture wassealed and then put into a pre-heated oil bath at 70° C. for 12 h. Theresultant mixture was dissolved in dichloromethane (5 mL) andprecipitated in hexane (200 mL). To remove unreactive monomers, theprecipitate was further dissolved in dichloromethane (5 mL) andre-precipitated in ethyl ether (200 mL) to yield purified the randomcopolymer as a waxy liquid. Yield: 78%. GPC (THF) M_(n): 24.7 K. PDI:1.6. ¹H NMR (400 MHz, CDCl₃) δ: 8.45, 7.68, 7.11, 3.80-3.42, 3.02,2.04-1.65, 1.24-0.87. The molar ratio between two blocks was determinedby integrating the methoxy proton in the polyethylene glycol unit andthe aromatic proton in the pyridine and found to be 3.1:6.9(PEO:PDSEMA).

Nanogel Preparation: The polymer (10 mg) was dissolved in water andplaced in a vessel pre-heated at 70° C. for 10 min. When the polymersolution turned turbid, a measured amount of DTT was added. Then themixture was stirred for another hour to allow for crosslinking. Theresulting nanogels were purified by dialysis using a 10,000 g/molmembrane.

Crosslinking density: To determine the crosslinking density, the polymer(0.8 mg/mL) in water was first treated with the requisite amount of DTT(10, 20, 50 mol % compared to PDS groups), and stir the mixtureovernight at room temperature. UV-vis measurements were performed withsamples of this solution diluted ten times. Once this was measured, theamount of pyridothione was calculated based on its known molarextinction coefficient (8.08×10³ M⁻¹cm⁻¹ at 343 nm) (Bioconjugate Chem.2006, 17, 1376-1384). The percentage of cross-linking was calculated byassuming that formation of a single, crosslinking disulfide bond wouldrequire cleavage of two PDS units and produce two pyridothionemolecules.

Nanogels with Encapsulated Lipophilic Guest Molecules: The polymer (10mg) and Nile red (2 mg) were dissolved in 200 μL of acetone and ameasured amount of DTT (2 μmol, 4 μmol and 5 μmol for 10 mol %, 20 mol %and 50 mol % respectively against PDS groups) was added. After stirringfor 10 min, 1 mL of deionized water was added and the mixture wasstirred overnight at room temperature, open to the atmosphere allowingthe organic solvent to evaporate. Excess insoluble Nile red was removedby filtration and pyridothione was removed from the nanogel solution byultrafiltration (triplicate) using a membrane with a molecular weightcutoff of 10,000 g/mol (Amicon Ultra cell-10K). For FRET experiments,nanogels were prepared using 1% DiO and 1% DiI in acetone instead ofNile red using the same procedure. Dox encapsulated nanogels wereprepared using doxorubicin hydrochloride dissolved in CH₂Cl₂ with 3 eq.NEt₃ using the same procedure.

Cell Culture: The cell viabilities of the nanogels were tested with 293Tcells. 293T cells were cultured in T75 cell culture flasks usingDulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with10% fetal bovine serum (FBS) supplement. The cells were seeded at 10,000cells/well/200 μL, in a 96 well plate and allowed to grow for 24 h underincubation at 37° C. and 5% CO₂. These cells were then treated withnanogels of different concentrations and were incubated for another 24h. Cell viability was measured using the Alamar Blue assay with eachdata point measured in triplicate. Fluorescence measurements were madeusing the plate reader SpectraMax M5 by setting the excitationwavelength at 560 nm and monitoring emission at 590 nm on a black wellplate. The toxicity of Dox-encapsulated polymer nanogels was testedagainst the MCF-7 cell line. The cells were treated withDox-encapsulated polymer nanogels of different concentrations and wereincubated for 72 h. Cell death was measured by the Alamar Blue assay intriplicate.

Laser Scanning Confocal Microscopy: The laser confocal experiment wasperformed with MCF-7 cells. MCF-7 cells were cultured in T75 cellculture flask containing DMEM/F12 with 10% FBS supplement. The cellswere seeded at 10,000 cells/100 μL in cover slip-bottomed Petri dishesand allowed to grow for 3 days at 37° C. in a 5% CO₂ incubator. Thecells in 2 mL of culture medium were treated with 200 μL of nanogelscontaining two dyes or doxorubicin and incubated at 37° C. for differenttime intervals before monitoring the cells by confocal microscopy.

Pentafluorophenyl Activated Esters and Bifunctional Cross-Linkers

Unless mentioned, all chemicals were used as received fromSigma-Aldrich. ¹H-NMR spectra were recorded on a 400 MHz Bruker NMRspectrometer while ¹⁹F-NMR spectra were collected on a 300 MHz BrukerNMR spectrometer. Molecular weight of the polymers was measured by gelpermeation chromatography (GPC, Waters) using a PMMA standard with arefractive index detector. THF was used as eluent with a flow rate of 1mL/min. DLS measurements were performed using a Malvern Nanozetasizer.FTIR spectra were recorded on a Perkin Elmer spectrometer.

Synthesis of pentafluorophenyl acrylate PFPA: Monomer was synthesized byusing a previously reported procedure. Briefly, pentafluorophenol (5.40g, 29.3 mmol) and 2,6-lutidine (3.80 mL, 32.7 mmol) were dissolved indry dichloromethane (50.0 mL). The above solution was cooled in an icebath and then acryloyl chloride was added (2.65 mL, 32.7 mmol). Afterstirring at ambient temperature for 12 h, the reaction mixture waswashed with water. The organic layer was collected, and dried overanhydrous sodium sulfate. Crude product was further purified by flashchromatograph to afford pure product. Yield: 54%. ¹H NMR (400MHz, CDCl₃)δ: 6.74 (d, 1H), 6.36 (q, 1H), 6.19 (d, 1H). ¹⁹F NMR (300 MHz, CDCl₃) δ:−152.5 (2F, d), −157.9 (1F, t), −162.3 (2F, d).

Synthesis of random copolymer PPFPA-r-PEGMA: To a Schlenk-flask,pentafluorophenyl acrylate (500.0 mg, 2.1 mmol), poly(ethylene glycol)methyl ether methacrylate (428.0 mg, 0.9 mmol), recrystallizedazodiisobutyonitrile (AIBN) (2.5 mg, 0.015 mmol), and4-cyano-4-((thiobenzoyl)-sulfanyl)pentanoicacid (33.5 mg, 0.120 mmol)were mixed in 1,4-dioxane (900 μL). The solution mixture was subjectedto three freeze-pump-thaw cycles. The sealed flask was immersed in apreheated oil bath at 75° C. The polymerization reaction was allowed toproceed for 5 days. The reaction was stopped by immersing the reactionflask in cold water. After the removal of 1,4-dioxane, the mixture wasprecipitated in hexane. The resulting mixture was dissolved in THF andthen precipitated in hexane. The same operation was repeated one moretime to afford the pure polymer. Yield 90%: ¹H NMR (400MHz, CDCl₃) δ:4.0-4.2 ppm, 3.5-3.8 ppm, 3.3-3.4 ppm, 2.7-3.2 ppm, 0.9-2.6 ppm. ¹⁹F NMR(300 MHz, CDCl₃) δ: −152 to −155 ppm (2F), −158 to −160 ppm (1F), −164to −166 ppm (2F). GPC (THF) Mn: 9.5 kDa. PDI: 1.3. By comparing theintegral of the methylene protons adjacent to the ester in thepolyethylene glycol unit and the polymer backbone proton in both thepolyethylene glycol and the pentafluorophenyl units, the molar ratio wasfound to be 3:7 (PEGMA:PFPA).

Nanogel preparation in THF: 4.0 mg of polymer was dissolved in a knownvolume of dry THF to make a polymer solution with the desiredconcentration. To the polymer solution was added 0.50 equivalents ofcross-linker with respected to the PFP groups and 1 equivalent ofdiisopropylethylamine (DIPEA). The solution was then heated at 50° C.for 4 h to afford 100% cross-linked nanogel. The cross-linking reactionwas characterized by FTIR. H₂O was added to the nanogel solution and theTHF was evaporated by stirring the solution in air for 24 h. The volumeof nanogel solution was adjusted by adding water to afford the desiredconcentration. Preparation of the nanogel loaded with DiI(DiIC18(3))follows the same procedure using a polymer solution mixed with 1wt %DiI. After cross-linking and evaporation of THF, the nanogel solutionwas further purified by triplicate dialysis in milliQ water for 3 days.

Nanogel preparation in water: 4.0 mg of polymer solution was dissolvedin H₂O to afford a polymer solution with the desired concentration. Tothe polymer solution, 0.50 equivalents of cross-linker with respect tothe PFP groups and 1 equivalent of diisopropylethylamine (DIPEA) wereadded. The polymer solution was heated at 50° C. for 4 hours to afford100% cross-linked nanogel.

Nanogel modification: 400 mL of 10.0 mg/mL polymer solution in THF washalf cross-linked by the addition of 0.25 equivalents of CYS and 0.50equivalents of DIPEA with respect to the PFP groups. After heating at50° C. for 4 hours, 2.0 equivalents of isopropylamine (IPA) orN,N-dimethylethylenediamine (DMEDA) with 2 equivalents of DIPEA (withrespect to the remaining PFP groups after cross-linking) were added tothe nanogel solution and heated at 50° C. for another 4 hours.Cross-linking and post-nanogel substitution were monitored by FTIR.Water was added to the modified nanogel solution. THF was evaporated bystirring the sample in air for 24 hours. The nanogel solution wasfurther purified by triplicate dialysis in milliQ water for 3 days.

Concurrent Binding and Delivery of Proteins and Lipophilic SmallMolecules

All chemicals, 2,2′-Dithiodipyridine, polyethylene glycol monomethylether methacrylate (MW 450), D,L-dithiothreitol (DTT),1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarbo-cyanine perchlorate(DiI), β-galactosidase (β-gal) from E. coli,2-nitrophenyl-β-D-galactopyranosidase, UltraPure Agarose and solventswere purchased from commercial sources and were used as received, unlessotherwise mentioned. Pyridyl disulfide ethyl methacrylate (PDSEMA) wasprepared using a previously reported procedure (Macromolecules 2006, 39,5595-5597). ¹H-NMR spectra were recorded on a 400 mHz Bruker NMRspectrometer using the residual proton resonance of the solvent as theinternal standard. Chemical shifts are reported in parts per million(ppm). Molecular weights of the polymers were estimated by gelpermeation chromatography (GPC) using PMMA standard with a refractiveindex detector. DLS measurements were performed using a MalvernNanozetasizer. UV-vis absorption spectra were recorded on a Varian(model EL 01125047) spectrophotometer. The fluorescence spectra wereobtained from a JASCO FP-6500 spectrofluorometer.

Synthesis of Random Copolymer: A mixture of 2-cyano-2-propylbenzodithioate (21 mg, 0.0949 mmol), PDSEMA (860 mg, 3.37 mmol),polyethylene glycol monomethyl ether methacrylate (620 mg, 1.31 mmol)and AIBN (5.0 mg, 0.0305 mmol) was dissolved in THF (3 mL) and degassedby performing three freeze-pump-thaw cycles. This reaction vessel wassealed and then placed in a pre-heated oil bath at 60° C. for 12 h. Toremove unreactive monomers and purify the polymer, the resultant mixturewas precipitated in cold ethyl ether (20 mL) to yield the randomcopolymer as a waxy solid. GPC (THF) M_(n): 23 K. PDI: 1.38. ¹H NMR (400MHz, CDCl₃) δ: 8.45, 7.66, 7.09, 4.20-4.06, 3.90-3.36, 3.01, 2.15-1.62,1.43-0.86. The molar ratio between two blocks was determined byintegrating the methoxy proton in the polyethylene glycol unit and thearomatic proton in the pyridine and found to be 29%:71% (PEO:PDSEMA)

Synthesis of CRRR peptide: 2-chloro-trityl chloride resin was selectedas the solid support to prepare the peptide using solid phase synthesis.Coupling reaction was carried out in the presence of 3 equiv. of Fmocprotected amino acid, 3 equiv. of HATU and 3 equiv. of DIPEA and wastested by Kaiser Test. Deprotection of Fmoc group was obtained bytreating the resin with 20% piperidine in DMF. The peptide was finallycleaved from resin by reacting with TFA/TIS/H₂O/EDT mixture.Precipitating the mixture in cold ether 5 times affords the crudepeptide. Peptide was used without further purification. Yield: 80%.Peptide was characterized by ¹H NMR and MS. ¹H NMR (400 MHz, D₂O) δ:4.45-4.52, 4.30-4.41, 4.03-4.11, 3.15-3.26, 2.92-3.01, 1.73-1.96,1.55-1.96. MS (FAB): exact mass calculated: 590.7. Found: 590.0.

Nanogel synthesis and surface modification: The polymer (10 mg) wasdissolved in water (1 mL) and the hydrophobic dye (0.2 mg of DiI) inacetone (100 μL) was added. The mixture was stirred for 6 h at roomtemperature, open to the atmosphere allowing the organic solvent toevaporate. To this micellar aggregate solution, was added a measuredamount of DTT and then the mixture was stirred for 12 h at roomtemperature to allow for crosslinking. To modify the nanogel's surface,the ligand (5 mg), CRRR, was added and then stirred for another 12 h.The resulting nanogels were purified by filtration and unattached excessligand was removed from the nanogel solution by ultrafiltration(triplicate) using a membrane with a molecular weight cutoff of 10,000g/mol (Amicon Ultra cell-10K).

Crosslinking density and peptide modification: In order to determine thecrosslinking density, UV-vis measurements were performed with samples ofthis solution diluted ten times. Once this was measured, the amount ofpyridothione was calculated based on its known molar extinctioncoefficient (8.08×10³ M⁻¹cm⁻¹ at 343 nm) (Bioconjugate Chem. 2006, 17,1376-1384). The percentage of cross-linking was calculated by assumingthat formation of a single, crosslinking disulfide bond would requirecleavage of two PDS units and produce two pyridothione molecules. Theattachment of the ligand was evaluated by further increase inpyridothione adsorption (FIG. 36).

Calculation of Crosslinking Density:

-   -   Sample concentration in UV: 0.1 mg/ml of polymer.    -   The molar ratio of each unit: PDS:PEG=x mol:y mol=71:29 (from        repeating unit by NMR)    -   PDS molecular weight=255 g/mol, PEG molecular weight=475 g/mol

-   So, x mol*255 g/mol+y mol*275 g/mol=0.1 mg

-   x mol=71/29 y mol

-   Therefore, x mole (PDS) is 2.23*10⁻⁷ mol in this solution    NG1

-   Absorbance is 0.31 at 343 nm.

-   By Beer's law,

-   A=εbc

-   0.31=8.08*10³ M⁻¹cm⁻¹*1 cm*c

-   c=3.84*10⁻⁵M

-   Therefore, 1 mL of resulting nanogel solution contains 3.84*10⁻⁸ mol    pyridothione (byproduct). It is 17 mol % of total PDS unit    (2.23*10⁻⁷ mol). It was assumed that two pyridothione are from one    disulfide formation and PDS unit is 71 mol % of total polymer.

-   Therefore, 17%/2*0.71=6% crosslinking density.    NG2

-   Absorbance is 0.703 at 343 nm.

-   By Beer's law,

-   A=εbc

-   0.703=8.08*10³ M⁻¹cm⁻¹*1 cm*c

-   c=8.70*10⁻⁵M

-   Therefore, 1 mL of resulting nanogel solution contains 8.70*10⁻⁸ mol    pyridothione (byproduct). It is 39 mol % of total PDS unit    (2.23*10⁻⁷ mol). It is assumed that two pyridothiones are from one    disulfide formation and PDS unit is 71 mol % of total polymer.

-   Therefore, 39%/2*0.71=14% crosslinking density.    Loading Degree of Hydrophobic Dye (DiI):

2 wt % DiI (0.2 mg/mL) were added into polymer solution (10 mg/mL). Thefinal loading amount was calculated based on molar extinctioncoefficient of DiI (104×10³ M⁻¹cm⁻¹) at 555 nm.

-   -   The polymer concentration: 0.1 mg/mL solution (diluted from 5        mg/mL stock from the nanogels used for UV measurement to 0.1        mg/ml to final volume of 0.6 mL)        NG1:

-   absorbance at 555 nm=0.310

-   C=0.310/(104×10³ M⁻¹cm¹×1 cm)=2.98×10⁻⁶M

-   Conversion to grams of DiI:

-   2.98 μM×961.32 g/mol×0.6 mL=1.7 μg DiI

-   wt % loading=DiI in nanogel/weight total nanogel=1.7 μg/0.1    mg×100=1.7 wt %

-   Loading efficiency: loaded amount/feed amount=1.7 μg/2 μg×100=85%    NG2:

-   absorbance at 555 nm=0.323

-   C=0.323/(104×10³ M⁻¹cm⁻¹×1 cm)=3.11×10⁻⁶M

-   Conversion to grams of DiI:

-   3.11 μM×961.32 g/mol×0.6 mL=1.8 μg DiI

-   wt % loading=DiI in nanogel/weight total nanogel=1.8 μg/0.1    mg×100=1.8 wt %

-   Loading efficiency: loaded amount/feed amount=1.8 μg/2 μg×100=90%

Labeling of β-gal with FITC: Fluorescein isothiocyanate isomer I (FITC)was dissolved in dimethyl sulfoxide at a concentration of 4 mg/mL. Theβ-gal (2.5 mg) was dissolved in 900 μL of 0.1 M sodium bicarbonatesolution (pH 9.0) and mixed with 250 μL freshly prepared FITC solution.The mixture was protected from light and stirred gently at roomtemperature for 2 h. The resulting FITC labeled β-gal was purified bysize exclusion chromatography with Sephadex G-25 as stationary phase andphosphate buffer (5 mM, pH 7.4) as mobile phase. Finally, the β-galconcentration and labeling efficiency were measured by UV-vis absorptionspectroscopy.

Nanogel-protein complex optimization: NG-CRRR with encapsulated DiI (2mg/mL) and FITC labeled β-gal (1 mg/mL) stock solutions were prepared in5 mM Sodium phosphate buffer. β-gal-NG-CRRR complex solutions wereprepared from 1:1 to 1:6 ratios (β-gal:NG-CRRR) and incubated for 1 hbefore spectroscopic measurements. The change of the intensity of FITC,which occurs by FRET between FITC in β-gal and DiI inside nanogels, wasrecorded by fluorescence spectroscopy with increasing nanogelconcentration at same β-gal concentration in the solution. The intensityof FITC was decreasing as increasing the DiI-containing nanogels andstopped changing for NG1-CRRR-β-gal complex at 2:1 ratio and at 4:1ratio for NG2-CRRR-β-gal, indicating that the stable complexation isformed above this concentration.

Estimation of Number of Nanogels Per Protein in Complex:

-   Diameter Values (DLS):-   β-gal=10 nm-   NG1-CRRR=20 nm NG1-CRRR-β-gal=40 nm-   NG2-CRRR=12 nm NG2-CRRR-β-gal=30 nm    Calculation for NG1:-   NG1-CRRR-β-gal complex radius=20 nm-   Volume=4/3π (20 nm)³=3.35×10⁴ nm³-   NG1-CRRR radius=10 nm-   Volume=4/3π (10 nm)³=4.19×10³ nm³-   β-gal radius=5 nm-   Volume=4/3π (5 nm)³=5.23×10² nm³-   Note: The ratio is 2:1 of NG1 and β-gal.-   Assuming that number of NG1-CRRR is 2p and number of β-gal is p in    complex.-   The summation of volume of 2p NG1-CRRR and p β-gal=the volume of    complex:-   2p×4.19×10³ nm³+p×b 5.23×10 ² nm³=3.35×10⁴ nm³-   p=3.35×10⁴ nm³/(2×4.19×10³ nm³+5.23×10² nm³)=3.76-   Thus, the average numbers of NG1-CRRR and β-gal per complex are 7.6    and 3.8 respectively.    Calculation for NG2:-   NG2-CRRR-β-gal complex radius=15 nm-   Volume=4/3π (15 nm)³=1.41×10⁴ nm³-   NG2-CRRR radius=6 nm.-   Volume=4/3π (6 nm)³=9.04×10² nm³-   β-gal radius=5 nm-   Volume=4/3π (5 nm)³=5.24×10² nm³-   Note: The ratio is 4:1 of NG2 and β-gal.-   Assuming that number of NG2 is 4q and number of (β-gal is q in    complex.-   The summation of volume of 4q NG2 and q β-gal=the volume of complex:-   4q×b 9.04×10 ² nm³+q×5.24×10² nm³=1.41×10⁴ nm³-   q=1.41×10⁴ nm³/(4×9.04×10² nm³+5.24×10² nm³)=3.41    Result:-   Thus, the average numbers of NG2-CRRR and β-gal per complex are 13.6    and 3.4 respectively.

Agarose Gel Electrophoresis Studies: Agarose gel electrophoresis wasperformed to observe nanogel and protein complexation taking advantageof the nanogel (positive) and β-gal (negative) charges differences. 10μL samples were prepared in Milli Q water: (1) β-gal control (100 μg),(2) NG1-CRRR control (200 μg), (3) NG1-CRRR: β-gal complex (200 μg: 100μg: (4) NG2-CRRR control (400 μg), and (5) NG2-CRRR: β-gal complex (400μg: 100 μg: with the addition of 1 μL Bromophenol blue (10 mg/mL). Thenanogels were incubated with the protein for 1 hour at room temperature.To prepare the gel, 270 mg of Ultra Pure Agarose was diluted in 1×TAEbuffer pH 8.0 and the solution was microwaved until getting ahomogeneous solution. The gel was poured in a FisherBiotechElectrophoresis System (FB-SB-710) and let stand until the gelsolidified with an 8 well comb. The gel was run with 1×TAE buffer at 100mV for 1 hour and 10 minutes at room temperature. The gel was washedwith Milli Q water for 10 minutes and stained with Gel Code Blue for 1hour.

As shown in FIG. 35 complexation of NG1-CRRR with β-gal is evident inLane 3. Free protein in lane 3 almost disappeared and there is someextension of the nanogel mark towards the cathode showing interactionbetween the nanogel and protein charges meaning that the positivecharges of the nanogel are shielded by negative charges of β-gal. Somecomplexation between NG2-CRRR and β-gal is observed as well, howeverthis interaction is weaker than that with NG1.

β-gal activity assay: Solutions of native β-gal (MW=116, 300 g/molmonomeric form), β-gal-NG1-CRRR (1:2 ratio) and β-gal-N2-CRRR (1:4ratio) were prepared in PBS buffer pH 7.4 and incubated for 1 hour.β-gal activity was assessed in a 96 well plate using SpectraMax M5 platereader. By adding the colorimetric substrate2-nitrophenyl-β-D-galactopyranoside (2.5 mM) activity of β-gal wasrecorded by the absorbance increase over time of pNP at 405 nmexcitation wavelength.

Laser Scanning Confocal Microscopy: The laser confocal experiment wasperformed with HeLa cells which were cultured in T75 flasks containingDMEM/F12 with 10% FBS supplement. The cells were seeded at 10,000cells/100 μL in cover slip-bottomed Petri dishes and allowed to grow for1 day at 37° C. in a 5% CO₂ incubator. The cells in 2 mL of culturemedium were treated with nanogels (0.1 mg/mL) containing dye and boundto protein; incubated for 3 hours at 37° C. before monitoring the cellsby confocal microscopy (excitation of both dye molecules at 488 nm and543 nm for FITC and DiI, respectively)

X-Gal Studies:

HeLa cells were seeded in a 96 well plate at 10,000 cells/well. After 24hours cells were washed with PBS. NG1 and NG2 complexes with β-gal wereprepared at ratios of 2:1 and 4:1 respectively and added to cells inserum free DMEM/F12 media. Cells were incubated with the complexes for 3hours. This was followed by washing cells with PBS twice and addition ofDMEM/F12 with 10% FBS supplement. After another 3 hours of incubation,X-gal staining studies were carried out according to the manufacturers'protocol (Genlantis). Briefly, after fixing cells for 15 minutes theywere washed twice with 1×PBS followed by addition of 80 μL 1× stainingbuffer. After overnight incubation cells were washed again and imageswere taken with an inverted microscope at 40× magnification using theMicron software.

Ligand-Decorated Nanogels and Cellular Targeting

General. 2,2′-Dithiodipyridine, 2-mercaptoethanol, polyethylene glycolmonomethyl ether methacrylate (MW 450), D,L-dithiothreitol (DTT),1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine perchlorate(DiI) and 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), folic acid,cyclo(Arg-Gly-Asp-D-Phe-Cys) peptide, 2-cyano-2-propyl benzodithioateand other conventional reagents were obtained from commercial sourcesand were used as received, unless otherwise mentioned. Polymers weresynthesized using RAFT polymerization and then purified byprecipitation. Pyridyl disulfide ethyl methacrylate (PDSEMA) wasprepared using a previously reported procedure. (Ghosh, et al. 2006Macromolecules 39, 5595-5597) ¹H-NMR spectra were recorded on a 400 MHzBruker NMR spectrometer using the residual proton resonance of thesolvent as the internal standard. Molecular weights of the polymers wereestimated by gel permeation chromatography (GPC) using PMMA standardwith a refractive index detector. DLS measurements were performed usinga Malvern Nanozetasizer. The fluorescence spectra were obtained from aJASCO FP-6500 spectrofluorimeter. Transmission electron microscopy (TEM)images were taken from JEOL 100CX at 100 KV.

Random copolymer. A mixture of 2-cyano-2-propyl benzodithioate (21.0 mg,0.095 mmol), PDSEMA (860 mg, 3.37 mmol), polyethylene glycol monomethylether methacrylate (620 mg, 1.31 mmol) and AIBN (5.0 mg, 0.031 mmol) wasdissolved in THF (3 mL) and degassed by performing threefreeze-pump-thaw cycles. This reaction vessel was sealed and then putinto a pre-heated oil bath at 60° C. for 12 h. To remove unreactivemonomers, the resultant mixture was precipitated in cold diethylether(20 mL) to yield the random copolymer as a waxy liquid. GPC (THF) M_(n):23 K. PDI: 1.38. ¹H NMR (400 MHz, CDCl₃) δ: 8.45, 7.66, 7.09, 4.20-4.06,3.90-3.36, 3.01, 2.15-1.62, 1.43-0.86. The molar ratio between twoblocks was determined by integrating the methoxy proton in thepolyethylene glycol unit and the aromatic proton in the pyridine andfound to be 29%:71% (PEO:PDSEMA).

Cell penetrating peptide CRRR: Peptide was synthesized on2-chloro-trityl chloride resin by solid phase synthesis using standardFmoc methodology. Briefly, coupling reaction was achieved by 3 equiv.Fmoc protected amino acid, 3 equiv. HATU, and 3 equiv DIPEA in DMF andmonitored by Kaiser Test. Fmoc protection group was removed by 20%piperidine in DMF. Cleavage of peptide from resin was performed in thepresence of TFA/TIS/H₂O/EDT mixture. Then cleavage mixture wasprecipitated in cold ether 5 times to afford crude peptide. Peptide wasused without further purification. Yield: 80%. Peptide was characterizedby ¹H-NMR and mass spectrometry.

Cysteine-folic acid: The ligand was prepared on cysteine preloaded2-chlorotrityl resin by solid phase synthesis. The resin was treatedwith 1.2 eq folic acid and 1.2 eq DIPEA in DMSO for 24 hours. Thecoupling reaction was repeated twice. Cleavage of cysteine-folic acidwas done by treating the resin with TFA/TIS/H₂O/EDT mixture. Crudeproduct was obtained by precipitation in cold ether 5 times. Yield: 78%.Characterization was followed by ¹H-NMR and mass spectrometry.

T-NGs preparation: The polymer (10 mg) was dissolved in water (1 mL) andhydrophobic dyes (0.1 mg of DiO or DiI) or 0.5 mg paclitaxel in acetone(100 μL) was added and the mixture was stirred for 6 h at roomtemperature, open to the atmosphere allowing the organic solvent toevaporate. It was placed in a vessel pre-heated at 60° C. for 10 min andthen a measured amount of DTT was added. Then the mixture was stirredfor 30 min to allow for crosslinking. To modify the nanogels surface,the ligand (5 mg), CRRR, cRGD, or cyteine-modified folic acid, was addedand then stirred for another 1 h. The resulting nanogels were purifiedby filtration and unattached excess ligand was removed from the nanogelsolution by ultrafiltration (triplicate) using a membrane with amolecular weight cutoff of 10,000 g/mol (Amicon Ultra cell-10K).

Crosslinking density and surface modification: In order to determine thecrosslinking density, the polymer in water was first treated with therequisite amount of DTT (20 mol % compared to PDS groups), and stir themixture at 60° C. UV-visible spectroscopic measurements were performedwith samples of this solution diluted ten times. Once this was measured,the amount of pyridothione was calculated based on its known molarextinction coefficient (8.08×10³ M⁻¹cm⁻¹ at 343 nm). (Kavimandan, et al.2006 Bioconjugate Chem., 17, 1376-1384) The percentage of cross-linkingwas calculated by assuming that formation of a single, crosslinkingdisulfide bond would require cleavage of two PDS units and produce twopyridothione molecules. The attachment of the ligand was evaluated byfurther increase in pyridothione absorption.

Cell culture: The cell viabilities of the nanogels were tested with 293Tcells. 293T cells were cultured in T75 cell culture flasks usingDulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12) with10% fetal bovine serum (FBS) supplement. The cells were seeded at 10,000cells/well/200 μL in a 96 well plate and allowed to grow for 24 hoursunder incubation at 37° C. and 5% CO₂. These cells were then treatedwith nanogels of different concentrations and were incubated for another24 hours. Cell viability was measured using the Alamar Blue assay witheach data point measured in triplicate. Fluorescence measurements weremade using the plate reader SpectraMax M5 by setting the excitationwavelength at 560 nm and monitoring emission at 590 nm on a black wellplate. The toxicity of paclitaxel (PTX)-encapsulated polymer aggregates,NG, and NG-RGD were tested against the MCF7 and HeLa cell line. Thecells were treated with PTX-encapsulated polymer nanogels of 10 μMconcentrations and were incubated for 72 hours. Cell death was measuredby the Alamar Blue assay in triplicate.

Laser scanning confocal microscopy: The laser confocal experiment wasperformed with different cell lines. Each cell line was cultured in T75cell culture flask containing DMEM/F12 with 10% FBS supplement. Thecells were seeded at 10,000 cells/100 μL in cover slip-bottomed petridishes and allowed to grow for 1 day at 37° C. in a 5% CO₂ incubator.The cells in 2 mL of culture medium were treated with polymeraggregates, nanogels, or T-NGs (0.1 mg/mL) containing dyes and incubatedat 37° C. for different time intervals before monitoring the cells byconfocal microscopy.

Calculation of the number of ligand (CRRR) per T-NG. The attachment ofthe ligand was evaluated by further increase in pyridothione absorption.Based on UV-vis measurement and molar extinction coefficient ofpyridothione, the amount of the ligand was calculated that is attachedonto the surface of nanogels. The number of ligand per T-NG wasestimated by following equation. It was assumed that the nanogel densityis around 1.00 g/cm³. The volume of one T-NG=4πr³/3=4π (21nm)³/3=3.879×10⁴ nm³=3.879×10⁻²³ m³

$\begin{matrix}\begin{matrix}{{{The}\mspace{14mu}{mass}\mspace{14mu}{of}\mspace{14mu}{one}\mspace{14mu} T\text{-}{NG}} = \frac{{the}\mspace{14mu}{volume}\mspace{14mu}{of}\mspace{14mu} T\text{-}{NG}}{{density}\mspace{14mu}{of}\mspace{14mu} T\text{-}{NG}}} \\{= {3.879 \times 10^{- 23}\mspace{14mu}{m^{3}/1.00}\mspace{14mu} g\text{/}{cm}^{3}}} \\{= {3.879 \times 10^{- 17}\mspace{14mu} g}}\end{matrix} \\\begin{matrix}{{{The}\mspace{14mu}{number}\mspace{14mu}{of}\mspace{14mu} T\text{-}{NG}\mspace{14mu}{in}\mspace{14mu}{solution}} = \frac{{total}\mspace{14mu}{nanogel}\mspace{14mu}{amount}}{{the}\mspace{14mu}{mass}\mspace{14mu}{of}\mspace{14mu}{one}\mspace{14mu} T\text{-}{NG}}} \\{= {6.35 \times 10^{- 5}\mspace{11mu}{g/3.879} \times 10^{- 17}\mspace{14mu} g}} \\{= {1.637 \times 10^{12}}}\end{matrix}\end{matrix}$The molar concentration of the ligand which is attached on the surfaceof nanogel is obtained by absorption intensity and extinctioncoefficient of pyridothione, which is 0.01925 mmol. Therefore, thenumber of ligand per one T-NG is estimated like following:

1.925 × 10⁻⁸  mol × 6.02 × 10²³  mol⁻¹/the  number  of  T-NG  in  solution = 7.08 × 10³

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance which can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

The invention claimed is:
 1. A nano-assembly for controlled delivery of a therapeutic or diagnostic agent to a biological site, comprising: a water-soluble polymer host comprising a crosslinked network of a random co-polymer having the structural formula:

wherein p is an integer from about 1 to about 20, the co-polymer has a molecular weight from about 1,000 to about 100,000, the ratio of i:j is in the range from about 2:8 to about 8:2, and from about 1% to about 95% of j is substituted by k, a guest molecule comprising a therapeutic, diagnostic, or imaging agent non-covalently associated with the polymer host, wherein the therapeutic or diagnostic agent is releasable upon partial or complete de-crosslinking of the crosslinked network of polymer molecules at or near the biological site, and a targeting moiety selected from an antibody, an aptamer, a peptide, and a small molecule ligand, covalently linked to or non-covalently associated with the polymer host.
 2. The nano-assembly of claim 1, wherein the crosslinked network of polymer molecules is crosslinked both inter-molecularly and intra-molecularly.
 3. The nano-assembly of claim 1, wherein the de-crosslinking of the crosslinked polymer molecules is due to a biological or chemical stimulus at the biological site.
 4. The nano-assembly of claim 3, wherein the stimulus is a pH value at the biological site.
 5. The nano-assembly of claim 1, wherein the stimulus is induced by an external light signal.
 6. The nano-assembly of claim 1, wherein the loading weight percentage of the guest molecule is from about 2% to about 70%.
 7. The nano-assembly of claim 6, wherein the crosslinked network of polymer molecules have a crosslinking density from about 5% to about 60%, relative to the total number of structural units in the polymer. 